Microfluidic nucleic acid analysis

ABSTRACT

Nucleic acid from cells and viruses sampled from a variety of environments may purified and expressed utilizing microfluidic techniques. Individual or small groups of cells or viruses may be isolated in microfluidic chambers by dilution, sorting, and/or segmentation. The isolated cells or viruses may be lysed directly in the microfluidic chamber, and the resulting nucleic acid purified by exposure to affinity beads. Subsequent elution of the purified nucleic acid may be followed by ligation and cell transformation, all within the same microfluidic chip. Cell isolation, lysis, and nucleic acid purification may be performed utilizing a highly parallelized microfluidic architecture to construct gDNA and cDNA libraries.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/406,451, filed on Jan. 13, 2017, which is a continuation of U.S.patent application Ser. No. 14/494,284, filed on Sep. 23, 2014 (now U.S.Pat. No. 9,579,650), which is a continuation of U.S. patent applicationSer. No. 10/678,946, filed on Oct. 2, 2003 (now U.S. Pat. No.8,871,446), which claims priority from U.S. provisional patentapplication No. 60/415,407, filed Oct. 2, 2002; U.S. provisional patentapplication No. 60/444,022, filed Jan. 31, 2003; U.S. provisional patentapplication No. 60/494,377, filed Aug. 11, 2003; and U.S. provisionalpatent application No. 60/494,388, filed Aug. 11, 2003. Each of thesepreviously filed patent applications is hereby incorporated herein byreference for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.BES-0119493 awarded by the National Science Foundation and under GrantNo. DAAD19-00-1-0392 awarded by DARPA. The government has certain rightsin the invention.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in file SequenceListing_087159-1031245.txtcreated on Dec. 2, 2016, 866 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference in its entiretyfor all purposes.

BACKGROUND OF THE INVENTION

There evidence that in many ecosystems more than 99% of the microbialpopulation resists laboratory cultivation. N. R. Pace, “A Molecular Viewof Microbial Diversity and the Biosphere”, Science 276, 734 (1997). Thishas led to the suggestion that microbial ecosystems in human beings aremore complex than previously suspected, and that some or many of theuncultivatable microbes may be infectious agents. D. A. Relman, “TheSearch for Unrecognized Pathogens”, Science 284, 1308 (1999).

A substantial number of illnesses resemble infectious disease, andunexplained death or critical illnesses occur in 0.5 persons per 100,000population. B. A. Perkins et al., “Unexplained Deaths Due to PossiblyInfectious Causes in the United States: Defining the Problem andDesigning Surveillance and Laboratory Approaches”, Emerging Inject. Dis.2, 47 (1996). Many such pathogens resist identification by traditionalphenotypic methods, which has led to the development of genotypicmethods of pathogen identification.

As a result, the last twenty years have seen a growing list ofconnections between human disease and previously unsuspected microbialpathogens. TABLE 1 lists examples of human disease and associatedmicrobial pathogens revealed during the past 20 years. All of theseinfectious agents, except Heliobacter pylori, were first identifieddirectly from clinical specimens using genotypic approaches.

TABLE 1 Disease Infectious Agent Peptic ulcer disease Heliobacter pyloriNon-A, non-B hepatitis Hepatitis C virus Bacillary angiomatosisBartonella henselae Whipple's disease Tropheryma whippelii Hantaviruspulmonary syndrome Sin Nombre virus Kaposi's sarcoma Kaposi'ssarcoma-associated herpesvirus

Although the human body offers a highly diverse ecosystem for bacteriaand viruses, it has been difficult to make precise measurements ofdiversity and population distribution because many organisms livingwithin it have proven difficult or impossible to culture.

Accordingly, there is a need in the art for techniques and apparatusesfor characterizing and analyzing bacteria and viruses existing in avariety of environments.

SUMMARY OF THE INVENTION

Nucleic acid from cells and viruses sampled from a variety ofenvironments may purified and expressed utilizing microfluidictechniques. In accordance with one embodiment of the present invention,individual or small groups of cells or viruses may be isolated inmicrofluidic chambers by dilution, sorting, and/or segmentation. Theisolated cells or viruses may be lysed directly in the microfluidicchamber, and the resulting nucleic acid purified by exposure to affinitybeads. Subsequent elution of the purified nucleic acid may be followedby ligation and cell transformation. In one specific application, cellisolation, lysis, and nucleic acid purification may be performedutilizing a highly parallelized microfluidic architecture to creategenomic and complementary DNA libraries.

An embodiment of a microfluidic device in accordance with the presentinvention comprises a first microfluidic chamber, and a firstmicrofluidic channel configured to deliver a sample containing at leastone of a virus, a bacterium, and a cell to the first microfluidicchamber. A second microfluidic flow channel is configured to deliver alysis chemical to the first microfluidic chamber.

An embodiment of a method in accordance with the present invention forpurifying nucleic acid, comprises, providing a sample comprising atleast one of bacteria, viruses, and cells. A subset of at least one ofbacteria, viruses, and cells is physically isolated within amicrofluidic chamber. The at least one of the bacteria, viruses, andcells is lysed within the microfluidic chamber. Nucleic acid is flowedfrom the at least one of the lysed bacteria, viruses, and cells througha second microfluidic chamber containing a nucleic acid purificationmedium.

An embodiment of a method in accordance with the present invention formicrofluidic processing, comprises, providing a sample in parallel to aplurality of microfluidic structures, and controlling processing of thesample by the plurality of microfluidic structures by manipulation of asingle control structure in common communication with the plurality ofmicrofluidic structures.

An embodiment of a method in accordance with the present invention foranalyzing a sample, comprises, flowing a sample comprising at least oneof viruses and cells down a microfluidic channel. Portions of themicrofluidic channel are isolated into segments containing a limitednumber of bacteria, viruses, or cells. At least one of genetic analysis,biochemical analysis, and cloning of genomic DNA or cDNA is performed onthe limited number of bacteria, viruses, or cells.

An embodiment of a method in accordance with the present invention forprocessing nucleic acid, comprises, providing a sample comprisingmultiple biological entities, and physically isolating an individualbiological entity or a subset of the biological entities within amicrofluidic chamber. The individual biological entity or the subset ofbiological entities is lysed, and experimentally manipulatable nucleicacid preparations are obtained from the individual biological entity orthe subset of biological entities.

An embodiment of a method in accordance with the present invention forobtaining nucleic acid from a sample of heterogenous biologicalelements, comprises, providing a sample comprising heterogenousbiological elements, and physically isolating an individual biologicalelement or a subset of the biological elements of the sample within amicrofluidic chamber. The individual biological element or the subset ofbiological elements is lysed, and nucleic acid from the individualbiological entity or the subset of biological entities is purified. Atleast a portion of the purified nucleic acid is amplified.

An embodiment of a method in accordance with the present invention forcharacterizing phylogenetic, gene, and functional diversity exhibited bya specific environment comprising a plurality of biological elements,comprises, providing a sample from the environment comprisingheterogenous biological elements. An individual biological element or asubset of the biological elements of the sample is physically isolatedwithin a microfluidic chamber. The individual biological element or thesubset of biological elements are lysed to expose nucleic acid presenttherein. The exposed nucleic acid from the individual biological entityor the subset of biological entities is purified. At least a portion ofthe purified nucleic acid is amplified, and at least one ofphylogenetic, gene, and functional diversity of the amplified portion ofthe purified nucleic acid is identified.

An embodiment of a method in accordance with the present invention forobtaining genetic information regarding an individual biological elementof a complex environmental sample, comprises, providing an environmentalsample comprising heterogenous biological elements, and physicallyisolating within a microfluidic chamber an individual biological elementor a subset of the biological elements of the sample. The individualbiological element or the subset of biological elements is lysed toexpose nucleic acid present therein. The exposed nucleic acid ispurified, and at least a portion of the purified nucleic acid isamplified.

These and other embodiments of the present invention, as well as itsadvantages and features, are described in more detail in conjunctionwith the text below and attached figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of a first elastomeric layer formed on top ofa micromachined mold.

FIG. 2 is an illustration of a second elastomeric layer formed on top ofa micromachined mold.

FIG. 3 is an illustration of the elastomeric layer of FIG. 2 removedfrom the micromachined mold and positioned over the top of theelastomeric layer of FIG. 1

FIG. 4 is an illustration corresponding to FIG. 3, but showing thesecond elastomeric layer positioned on top of the first elastomericlayer.

FIG. 5 is an illustration corresponding to FIG. 4, but showing the firstand second elastomeric layers bonded together.

FIG. 6 is an illustration corresponding to FIG. 5, but showing the firstmicromachined mold removed and a planar substrate positioned in itsplace.

FIG. 7A is an illustration corresponding to FIG. 6, but showing theelastomeric structure sealed onto the planar substrate.

FIG. 7B is a front sectional view corresponding to FIG. 7A, showing anopen flow channel.

FIGS. 7C-7G are illustrations showing steps of a method for forming anelastomeric structure having a membrane formed from a separateelastomeric layer.

FIG. 7H is a front sectional view showing the valve of FIG. 7B in anactuated state.

FIGS. 8A and 8B illustrates valve opening vs. applied pressure forvarious flow channels.

FIG. 9 illustrates time response of a 100 μm×100 μm×10 μm RTVmicrovalve.

FIG. 10 is a front sectional view of the valve of FIG. 7B showingactuation of the membrane.

FIG. 11 is a front sectional view of an alternative embodiment of avalve having a flow channel with a curved upper surface.

FIG. 12A is a top schematic view of an on/off valve.

FIG. 12B is a sectional elevation view along line 23B-23B in FIG. 12A

FIG. 13A is a top schematic view of a peristaltic pumping system.

FIG. 13B is a sectional elevation view along line 24B-24B in FIG. 13A

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for an embodiment of the peristaltic pumping system of FIG.13.

FIG. 15A is a top schematic view of one control line actuating multipleflow lines simultaneously.

FIG. 15B is a sectional elevation view along line 26B-26B in FIG. 15A

FIG. 16 is a schematic illustration of a multiplexed system adapted topermit flow through various channels.

FIGS. 17A to 17D show plan views of one embodiment of a switchable flowarray.

FIGS. 18A to 18D show plan views of one embodiment of a cell pen arraystructure.

FIGS. 19A and 19B show plan and cross-sectional views illustratingoperation of one embodiment of a cell cage structure in accordance withthe present invention.

FIGS. 20A to 20D show plan views of operation of a structure utilizingcross-channel injection in accordance with the embodiment of the presentinvention.

FIG. 21 shows a plan view of one embodiment of a rotary mixing structurein accordance with the present invention.

FIG. 22A shows a simplified plan view illustrating a binary treemicrofluidic multiplexor operational diagram.

FIG. 22B shows a simplified plan view illustrating a tertiary treemicrofluidic multiplexor operational diagram.

FIG. 22C shows a simplified cross-sectional view of the generalmicrofluidic architecture of the devices of FIGS. 22A-B.

FIG. 23 plots the number of control lines versus the number of flowlines being controlled, for multiplexors of various base numbers.

FIG. 24 shows a simplified plan view of an embodiment of a microfluidicstructure utilizing control channels to control other control channels.

FIG. 24A shows a simplified cross-sectional view of the structure ofFIG. 24 taken along the line A-A′

FIG. 24B shows a simplified cross-sectional view of the structure ofFIG. 24 taken along the line B-B′.

FIG. 25 shows a simplified cross-sectional view of the generalmicrofluidic architecture of the device of FIGS. 24-24B.

FIG. 26 shows a simplified plan view of an alternative embodiment of amicrofluidic structure utilizing control channels to control othercontrol channels.

FIG. 26A shows a simplified cross-sectional view of the structure ofFIG. 26 taken along the line A-A′.

FIG. 26B shows a simplified cross-sectional view of the structure ofFIG. 26 taken along the line B-B′.

FIG. 27A shows an optical micrograph of a microfluidic comparator chip.

FIG. 27B is a simplified schematic view of the microfluidic comparatorchip of FIG. 27A.

FIGS. 27C to 27H are enlarged simplified plan views showing loading ofthe chamber of the microfluidic structure of FIG. 27A.

FIG. 28 is a simplified flow chart illustrating steps of manipulatingnucleic acids in accordance with embodiments of the present invention.

FIG. 29A shows a plan view of one embodiment of a microfluidicarchitecture in accordance with the present invention allowing celllysis and purification of DNA present therein.

FIGS. 29B to 29D show photographs of enlarged regions of themicrofluidic architecture of FIG. 22A.

FIGS. 30A and 30B show photographs of enlarged regions of themicrofluidic architecture of FIG. 29A before and after cell lysis.

FIGS. 31A and 31B show electrophoresis of nucleic acid samples purifiedin accordance with embodiments of the present invention.

FIGS. 32A and 32B show the results of analyzing different samples ofpurified nucleic material utilizing the microfluidic architecture ofFIG. 29A.

FIG. 33 shows a plan view of one embodiment of a microfluidicarchitecture in accordance with the present invention allowing ligationof purified nucleic acid, and transformation of cells with the ligatednucleic acid.

FIG. 33A shows an enlarged view of the region of the microfluidicarchitecture of FIG. 33 allowing ligation of purified nucleic acid.

FIG. 33B shows an enlarged view of the region of the microfluidicarchitecture of FIG. 33 allowing cell transformation with the ligatedpurified nucleic acid.

FIG. 34 shows an exploded view of a microfluidic structure forperforming PCR.

FIG. 35 plots fluorescence versus time for nucleic acids amplified byPCR utilizing the microfluidic structure shown in FIG. 34.

FIG. 36A shows a plan view of a matrix PCR architecture.

FIG. 36B shows an enlarged view of one cell of the matrix PCRarchitecture of FIG. 36A.

FIG. 37A is a plan view of an alternative microfluidic architecture inaccordance with the present invention

FIG. 37B is a photograph showing an enlargement of the adjacent lysisand cell chambers of FIG. 37A.

FIG. 37C is a photograph showing an enlargement of the adjacent bead andcell chambers of FIG. 37A.

FIGS. 37D to 37F show photographs of an enlarged portion of the cellchamber before, during, and after diffusive mixing of lysis chemistry,respectively.

FIG. 38 shows electrophoresis results for a series of experimentsperformed in order to demonstrate single cell sensitivity together withnegative controls.

FIG. 39 plots RT PCR products for both β-actin and ozf analyzed on a 2%agarose gel.

FIG. 40 shows a plan view of an alternative embodiment of a microfluidicarchitecture in accordance with the present invention.

FIGS. 41A to 41B show flow cytometric analysis of the surface markerexpression profile of murine bone marrow cells.

FIG. 42 illustrates a proposed regulatory network for murine pluripotenthematopoietic stem cells

DETAILED DESCRIPTION OF THE INVENTION

I. Microfabrication Overview

The following discussion relates to formation of microfabricated fluidicdevices utilizing elastomer materials, as described generally in U.S.patent application Ser. No. 10/10/679,997 (now U.S. Pat. No. 7,143,785),filed Sep. 24, 2003), Ser. No. 10/265,473 filed Oct. 4, 2002, Ser. No.10/118,466 filed Apr. 5, 2002, Ser. No. 09/826,585 filed Apr. 6, 2001,Ser. No. 09/724,784 filed Nov. 28, 2000, and Ser. No. 09/605,520, filedJun. 27, 2000. These previously-filed patent applications are herebyincorporated by reference for all purposes.

1. Methods of Fabricating

Exemplary methods of fabricating the present invention are providedherein. It is to be understood that the present invention is not limitedto fabrication by one or the other of these methods. Rather, othersuitable methods of fabricating the present microstructures, includingmodifying the present methods, are also contemplated.

FIGS. 1 to 7B illustrate sequential steps of a first preferred method offabricating the present microstructure, (which may be used as a pump orvalve). FIGS. 8 to 18 illustrate sequential steps of a second preferredmethod of fabricating the present microstructure, (which also may beused as a pump or valve).

As will be explained, the preferred method of FIGS. 1 to 7B involvesusing pre-cured elastomer layers which are assembled and bonded. In analternative method, each layer of elastomer may be cured “in place”. Inthe following description “channel” refers to a recess in theelastomeric structure which can contain a flow of fluid or gas.

Referring to FIG. 1, a first micro-machined mold 10 is provided.Micro-machined mold 10 may be fabricated by a number of conventionalsilicon processing methods, including but not limited tophotolithography, ion-milling, and electron beam lithography.

As can be seen, micro-machined mold 10 has a raised line or protrusion11 extending therealong. A first elastomeric layer 20 is cast on top ofmold 10 such that a first recess 21 will be formed in the bottom surfaceof elastomeric layer 20, (recess 21 corresponding in dimension toprotrusion 11), as shown.

As can be seen in FIG. 2, a second micro-machined mold 12 having araised protrusion 13 extending therealong is also provided. A secondelastomeric layer 22 is cast on top of mold 12, as shown, such that arecess 23 will be formed in its bottom surface corresponding to thedimensions of protrusion 13.

As can be seen in the sequential steps illustrated in FIGS. 3 and 4,second elastomeric layer 22 is then removed from mold 12 and placed ontop of first elastomeric layer 20. As can be seen, recess 23 extendingalong the bottom surface of second elastomeric layer 22 will form a flowchannel 32.

Referring to FIG. 5, the separate first and second elastomeric layers 20and 22 (FIG. 4) are then bonded together to form an integrated (i.e.:monolithic) elastomeric structure 24.

As can been seen in the sequential step of FIGS. 6 and 7A, elastomericstructure 24 is then removed from mold 10 and positioned on top of aplanar substrate 14. As can be seen in FIGS. 7A and 7B, when elastomericstructure 24 has been sealed at its bottom surface to planar substrate14, recess 21 will form a flow channel 30.

The present elastomeric structures form a reversible hermetic seal withnearly any smooth planar substrate. An advantage to forming a seal thisway is that the elastomeric structures may be peeled up, washed, andre-used. In preferred aspects, planar substrate 14 is glass. A furtheradvantage of using glass is that glass is transparent, allowing opticalinterrogation of elastomer channels and reservoirs. Alternatively, theelastomeric structure may be bonded onto a flat elastomer layer by thesame method as described above, forming a permanent and high-strengthbond. This may prove advantageous when higher back pressures are used.

As can be seen in FIGS. 7A and 7B, flow channels 30 and 32 arepreferably disposed at an angle to one another with a small membrane 25of substrate 24 separating the top of flow channel 30 from the bottom offlow channel 32.

In preferred aspects, planar substrate 14 is glass. An advantage ofusing glass is that the present elastomeric structures may be peeled up,washed and reused. A further advantage of using glass is that opticalsensing may be employed. Alternatively, planar substrate 14 may be anelastomer itself, which may prove advantageous when higher backpressures are used.

The method of fabrication just described may be varied to form astructure having a membrane composed of an elastomeric materialdifferent than that forming the walls of the channels of the device.This variant fabrication method is illustrated in FIGS. 7C-7G.

Referring to FIG. 7C, a first micro-machined mold 10 is provided.Micro-machined mold 10 has a raised line or protrusion 11 extendingtherealong. In FIG. 7D, first elastomeric layer 20 is cast on top offirst micro-machined mold 10 such that the top of the first elastomericlayer 20 is flush with the top of raised line or protrusion 11. This maybe accomplished by carefully controlling the volume of elastomericmaterial spun onto mold 10 relative to the known height of raised line11. Alternatively, the desired shape could be formed by injectionmolding.

In FIG. 7E, second micro-machined mold 12 having a raised protrusion 13extending therealong is also provided. Second elastomeric layer 22 iscast on top of second mold 12 as shown, such that recess 23 is formed inits bottom surface corresponding to the dimensions of protrusion 13.

In FIG. 7F, second elastomeric layer 22 is removed from mold 12 andplaced on top of third elastomeric layer 222. Second elastomeric layer22 is bonded to third elastomeric layer 20 to form integral elastomericblock 224 using techniques described in detail below. At this point inthe process, recess 23 formerly occupied by raised line 13 will formflow channel 23.

In FIG. 7G, elastomeric block 224 is placed on top of firstmicro-machined mold 10 and first elastomeric layer 20. Elastomeric blockand first elastomeric layer 20 are then bonded together to form anintegrated (i.e.: monolithic) elastomeric structure 24 having a membranecomposed of a separate elastomeric layer 222.

When elastomeric structure 24 has been sealed at its bottom surface to aplanar substrate in the manner described above in connection with FIG.7A, the recess formerly occupied by raised line 11 will form flowchannel 30.

The variant fabrication method illustrated above in conjunction withFIGS. 7C-7G offers the advantage of permitting the membrane portion tobe composed of a separate material than the elastomeric material of theremainder of the structure. This is important because the thickness andelastic properties of the membrane play a key role in operation of thedevice. Moreover, this method allows the separate elastomer layer toreadily be subjected to conditioning prior to incorporation into theelastomer structure. As discussed in detail below, examples ofpotentially desirable condition include the introduction of magnetic orelectrically conducting species to permit actuation of the membrane,and/or the introduction of dopant into the membrane in order to alterits elasticity.

While the above method is illustrated in connection with forming variousshaped elastomeric layers formed by replication molding on top of amicromachined mold, the present invention is not limited to thistechnique. Other techniques could be employed to form the individuallayers of shaped elastomeric material that are to be bonded together.For example, a shaped layer of elastomeric material could be formed bylaser cutting or injection molding, or by methods utilizing chemicaletching and/or sacrificial materials as discussed below in conjunctionwith the second exemplary method.

An alternative method fabricates a patterned elastomer structureutilizing development of photoresist encapsulated within elastomermaterial. However, the methods in accordance with the present inventionare not limited to utilizing photoresist. Other materials such as metalscould also serve as sacrificial materials to be removed selective to thesurrounding elastomer material, and the method would remain within thescope of the present invention. For example, gold metal may be etchedselective to RTV 615 elastomer utilizing the appropriate chemicalmixture.

2. Layer and Channel Dimensions

Microfabricated refers to the size of features of an elastomericstructure fabricated in accordance with an embodiment of the presentinvention. In general, variation in at least one dimension ofmicrofabricated structures is controlled to the micron level, with atleast one dimension being microscopic (i.e. below 1000 μm).Microfabrication typically involves semiconductor or MEMS fabricationtechniques such as photolithography and spincoating that are designedfor to produce feature dimensions on the microscopic level, with atleast some of the dimension of the microfabricated structure requiring amicroscope to reasonably resolve/image the structure.

In preferred aspects, flow channels 30, 32, 60 and 62 preferably havewidth-to-depth ratios of about 10:1. A non-exclusive list of otherranges of width-to-depth ratios in accordance with embodiments of thepresent invention is 0.1:1 to 100:1, more preferably 1:1 to 50:1, morepreferably 2:1 to 20:1, and most preferably 3:1 to 15:1. In an exemplaryaspect, flow channels 30, 32, 60 and 62 have widths of about 1 to 1000microns. A non-exclusive list of other ranges of widths of flow channelsin accordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 1000 microns, more preferably 0.2 to500 microns, more preferably 1 to 250 microns, and most preferably 10 to200 microns. Exemplary channel widths include 0.1 μm, 1 μm, 2 μm, 5 μm,10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm,110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm,200 μm, 210 μm, 220 μm, 230 μm, 240 μm, and 250 μm.

Flow channels 30, 32, 60, and 62 have depths of about 1 to 100 microns.A non-exclusive list of other ranges of depths of flow channels inaccordance with embodiments of the present invention is 0.01 to 1000microns, more preferably 0.05 to 500 microns, more preferably 0.2 to 250microns, and more preferably 1 to 100 microns, more preferably 2 to 20microns, and most preferably 5 to 10 microns. Exemplary channel depthsinclude including 0.01 μm, 0.02 μm, 0.05 μm, 0.1 μm, 0.2 μm, 0.5 μm, 1μm, 2 μm, 3 μm, 4 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm, 15 μm, 17.5 μm, 20μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100 μm, 150 μm, 200 μm,and 250 μm.

The flow channels are not limited to these specific dimension ranges andexamples given above, and may vary in width in order to affect themagnitude of force required to deflect the membrane as discussed atlength below in conjunction with FIG. 27. For example, extremely narrowflow channels having a width on the order of 0.01 μm may be useful inoptical and other applications, as discussed in detail below.Elastomeric structures which include portions having channels of evengreater width than described above are also contemplated by the presentinvention, and examples of applications of utilizing such wider flowchannels include fluid reservoir and mixing channel structures.

The Elastomeric layers may be cast thick for mechanical stability. In anexemplary embodiment, elastomeric layer 22 of FIG. 1 is 50 microns toseveral centimeters thick, and more preferably approximately 4 mm thick.A non-exclusive list of ranges of thickness of the elastomer layer inaccordance with other embodiments of the present invention is betweenabout 0.1 micron to 10 cm, 1 micron to 5 cm, 10 microns to 2 cm, 100microns to 10 mm.

Accordingly, membrane 25 of FIG. 7B separating flow channels 30 and 32has a typical thickness of between about 0.01 and 1000 microns, morepreferably 0.05 to 500 microns, more preferably 0.2 to 250, morepreferably 1 to 100 microns, more preferably 2 to 50 microns, and mostpreferably 5 to 40 microns. As such, the thickness of elastomeric layer22 is about 100 times the thickness of elastomeric layer 20. Exemplarymembrane thicknesses include 0.01 μm, 0.02 μm, 0.03 μm, 0.05 μm, 0.1 μm,0.2 μm, 0.3 μm, 0.5 μm, 1 μm, 2 μm, 3 μm, 5 μm, 7.5 μm, 10 μm, 12.5 μm,15 μm, 17.5 μm, 20 μm, 22.5 μm, 25 μm, 30 μm, 40 μm, 50 μm, 75 μm, 100μm, 150 μm, 200 μm, 250 μm, 300 μm, 400 μm, 500 μm, 750 μm, and 1000 μm.

3. Soft Lithographic Bonding

Preferably, elastomeric layers are bonded together chemically, usingchemistry that is intrinsic to the polymers comprising the patternedelastomer layers. Most preferably, the bonding comprises two component“addition cure” bonding.

In a preferred aspect, the various layers of elastomer are boundtogether in a heterogenous bonding in which the layers have a differentchemistry. Alternatively, a homogenous bonding may be used in which alllayers would be of the same chemistry. Thirdly, the respective elastomerlayers may optionally be glued together by an adhesive instead. In afourth aspect, the elastomeric layers may be thermoset elastomers bondedtogether by heating.

In one aspect of homogeneous bonding, the elastomeric layers arecomposed of the same elastomer material, with the same chemical entityin one layer reacting with the same chemical entity in the other layerto bond the layers together. In one embodiment, bonding between polymerchains of like elastomer layers may result from activation of acrosslinking agent due to light, heat, or chemical reaction with aseparate chemical species.

Alternatively in a heterogeneous aspect, the elastomeric layers arecomposed of different elastomeric materials, with a first chemicalentity in one layer reacting with a second chemical entity in anotherlayer. In one exemplary heterogenous aspect, the bonding process used tobind respective elastomeric layers together may comprise bondingtogether two layers of RTV 615 silicone. RTV 615 silicone is a two-partaddition-cure silicone rubber. Part A contains vinyl groups andcatalyst; part B contains silicon hydride (Si—H) groups. Theconventional ratio for RTV 615 is 10A:1B. For bonding, one layer may bemade with 30A:1B (i.e. excess vinyl groups) and the other with 3A:1B(i.e. excess Si—H groups). Each layer is cured separately. When the twolayers are brought into contact and heated at elevated temperature, theybond irreversibly forming a monolithic elastomeric substrate.

In an exemplary aspect of the present invention, elastomeric structuresare formed utilizing Sylgard 182, 184 or 186, or aliphatic urethanediacrylates such as (but not limited to) Ebecryl 270 or Irr 245 from UCBChemical.

In one embodiment in accordance with the present invention, two-layerelastomeric structures were fabricated from pure acrylated Urethane Ebe270. A thin bottom layer was spin coated at 8000 rpm for 15 seconds at170° C. The top and bottom layers were initially cured under ultravioletlight for 10 minutes under nitrogen utilizing a Model ELC 500 devicemanufactured by Electrolite corporation. The assembled layers were thencured for an additional 30 minutes. Reaction was catalyzed by a 0.5%vol/vol mixture of Irgacure 500 manufactured by Ciba-Geigy Chemicals.The resulting elastomeric material exhibited moderate elasticity andadhesion to glass.

In another embodiment in accordance with the present invention,two-layer elastomeric structures were fabricated from a combination of25% Ebe 270/50% Irr245/25% isopropyl alcohol for a thin bottom layer,and pure acrylated Urethane Ebe 270 as a top layer. The thin bottomlayer was initially cured for 5 min, and the top layer initially curedfor 10 minutes, under ultraviolet light under nitrogen utilizing a ModelELC 500 device manufactured by Electrolite corporation. The assembledlayers were then cured for an additional 30 minutes. Reaction wascatalyzed by a 0.5% vol/vol mixture of Irgacure 500 manufactured byCiba-Geigy Chemicals. The resulting elastomeric material exhibitedmoderate elasticity and adhered to glass.

Alternatively, other bonding methods may be used, including activatingthe elastomer surface, for example by plasma exposure, so that theelastomer layers/substrate will bond when placed in contact. Forexample, one possible approach to bonding together elastomer layerscomposed of the same material is set forth by Duffy et al, “RapidPrototyping of Microfluidic Systems in Poly (dimethylsiloxane)”,Analytical Chemistry (1998), 70, 4974-4984, incorporated herein byreference. This paper discusses that exposing polydimethylsiloxane(PDMS) layers to oxygen plasma causes oxidation of the surface, withirreversible bonding occurring when the two oxidized layers are placedinto contact.

Yet another approach to bonding together successive layers of elastomeris to utilize the adhesive properties of uncured elastomer.Specifically, a thin layer of uncured elastomer such as RTV 615 isapplied on top of a first cured elastomeric layer. Next, a second curedelastomeric layer is placed on top of the uncured elastomeric layer. Thethin middle layer of uncured elastomer is then cured to produce amonolithic elastomeric structure. Alternatively, uncured elastomer canbe applied to the bottom of a first cured elastomer layer, with thefirst cured elastomer layer placed on top of a second cured elastomerlayer. Curing the middle thin elastomer layer again results in formationof a monolithic elastomeric structure.

Where encapsulation of sacrificial layers is employed to fabricate theelastomer structure, bonding of successive elastomeric layers may beaccomplished by pouring uncured elastomer over a previously curedelastomeric layer and any sacrificial material patterned thereupon.Bonding between elastomer layers occurs due to interpenetration andreaction of the polymer chains of an uncured elastomer layer with thepolymer chains of a cured elastomer layer. Subsequent curing of theelastomeric layer will create a bond between the elastomeric layers andcreate a monolithic elastomeric structure.

Referring to the first method of FIGS. 1 to 7B, first elastomeric layer20 may be created by spin-coating an RTV mixture on microfabricated mold12 at 2000 rpm's for 30 seconds yielding a thickness of approximately 40microns. Second elastomeric layer 22 may be created by spin-coating anRTV mixture on microfabricated mold 11. Both layers 20 and 22 may beseparately baked or cured at about 80° C. for 1.5 hours. The secondelastomeric layer 22 may be bonded onto first elastomeric layer 20 atabout 80° C. for about 1.5 hours.

Micromachined molds 10 and 12 may be patterned photoresist on siliconwafers. In an exemplary aspect, a Shipley SJR 5740 photoresist was spunat 2000 rpm patterned with a high resolution transparency film as a maskand then developed yielding an inverse channel of approximately 10microns in height. When baked at approximately 200° C. for about 30minutes, the photoresist reflows and the inverse channels becomerounded. In preferred aspects, the molds may be treated withtrimethylchlorosilane (TMCS) vapor for about a minute before each use inorder to prevent adhesion of silicone rubber.

4. Suitable Elastomeric Materials

Allcock et al, Contemporary Polymer Chemistry, 2^(nd) Ed. describeselastomers in general as polymers existing at a temperature betweentheir glass transition temperature and liquefaction temperature.Elastomeric materials exhibit elastic properties because the polymerchains readily undergo torsional motion to permit uncoiling of thebackbone chains in response to a force, with the backbone chainsrecoiling to assume the prior shape in the absence of the force. Ingeneral, elastomers deform when force is applied, but then return totheir original shape when the force is removed. The elasticity exhibitedby elastomeric materials may be characterized by a Young's modulus.Elastomeric materials having a Young's modulus of between about 1 Pa-1TPa, more preferably between about 10 Pa-100 GPa, more preferablybetween about 20 Pa-1 GPa, more preferably between about 50 Pa-10 MPa,and more preferably between about 100 Pa-1 MPa are useful in accordancewith the present invention, although elastomeric materials having aYoung's modulus outside of these ranges could also be utilized dependingupon the needs of a particular application.

The systems of the present invention may be fabricated from a widevariety of elastomers. In an exemplary aspect, the elastomeric layersmay preferably be fabricated from silicone rubber. However, othersuitable elastomers may also be used.

In an exemplary aspect of the present invention, the present systems arefabricated from an elastomeric polymer such as GE RTV 615 (formulation),a vinyl-silane crosslinked (type) silicone elastomer (family). However,the present systems are not limited to this one formulation, type oreven this family of polymer; rather, nearly any elastomeric polymer issuitable. An important requirement for the preferred method offabrication of the present microvalves is the ability to bond multiplelayers of elastomers together. In the case of multilayer softlithography, layers of elastomer are cured separately and then bondedtogether. This scheme requires that cured layers possess sufficientreactivity to bond together. Either the layers may be of the same type,and are capable of bonding to themselves, or they may be of twodifferent types, and are capable of bonding to each other. Otherpossibilities include the use an adhesive between layers and the use ofthermoset elastomers.

Given the tremendous diversity of polymer chemistries, precursors,synthetic methods, reaction conditions, and potential additives, thereare a huge number of possible elastomer systems that could be used tomake monolithic elastomeric microvalves and pumps. Variations in thematerials used will most likely be driven by the need for particularmaterial properties, i.e. solvent resistance, stiffness, gaspermeability, or temperature stability.

There are many, many types of elastomeric polymers. A brief descriptionof the most common classes of elastomers is presented here, with theintent of showing that even with relatively “standard” polymers, manypossibilities for bonding exist. Common elastomeric polymers includepolyisoprene, polybutadiene, polychloroprene, polyisobutylene,poly(styrene-butadiene-styrene), the polyurethanes, and silicones.

Polyisoprene, Polybutadiene, Polychloroprene:

Polyisoprene, polybutadiene, and polychloroprene are all polymerizedfrom diene monomers, and therefore have one double bond per monomer whenpolymerized. This double bond allows the polymers to be converted toelastomers by vulcanization (essentially, sulfur is used to formcrosslinks between the double bonds by heating). This would easily allowhomogeneous multilayer soft lithography by incomplete vulcanization ofthe layers to be bonded; photoresist encapsulation would be possible bya similar mechanism.

Polyisobutylene:

Pure Polyisobutylene has no double bonds, but is crosslinked to use asan elastomer by including a small amount (˜1%) of isoprene in thepolymerization. The isoprene monomers give pendant double bonds on thePolyisobutylene backbone, which may then be vulcanized as above.

Poly(Styrene-Butadiene-Styrene):

Poly(styrene-butadiene-styrene) is produced by living anionicpolymerization (that is, there is no natural chain-terminating step inthe reaction), so “live” polymer ends can exist in the cured polymer.This makes it a natural candidate for the present photoresistencapsulation system (where there will be plenty of unreacted monomer inthe liquid layer poured on top of the cured layer). Incomplete curingwould allow homogeneous multilayer soft lithography (A to A bonding).The chemistry also facilitates making one layer with extra butadiene(“A”) and coupling agent and the other layer (“B”) with a butadienedeficit (for heterogeneous multilayer soft lithography). SBS is a“thermoset elastomer”, meaning that above a certain temperature it meltsand becomes plastic (as opposed to elastic); reducing the temperatureyields the elastomer again. Thus, layers can be bonded together byheating.

Polyurethanes:

Polyurethanes are produced from di-isocyanates (A-A) and di-alcohols ordi-amines (B-B); since there are a large variety of di-isocyanates anddi-alcohols/amines, the number of different types of polyurethanes ishuge. The A vs. B nature of the polymers, however, would make themuseful for heterogeneous multilayer soft lithography just as RTV 615 is:by using excess A-A in one layer and excess B-B in the other layer.

Silicones:

Silicone polymers probably have the greatest structural variety, andalmost certainly have the greatest number of commercially availableformulations. The vinyl-to-(Si—H) crosslinking of RTV 615 (which allowsboth heterogeneous multilayer soft lithography and photoresistencapsulation) has already been discussed, but this is only one ofseveral crosslinking methods used in silicone polymer chemistry.

5. Operation of Device

FIGS. 7B and 7H together show the closing of a first flow channel bypressurizing a second flow channel, with FIG. 7B (a front sectional viewcutting through flow channel 32 in corresponding FIG. 7A), showing anopen first flow channel 30; with FIG. 7H showing first flow channel 30closed by pressurization of the second flow channel 32.

Referring to FIG. 7B, first flow channel 30 and second flow channel 32are shown. Membrane 25 separates the flow channels, forming the top offirst flow channel 30 and the bottom of second flow channel 32. As canbe seen, flow channel 30 is “open”.

As can be seen in FIG. 7H, pressurization of flow channel 32 (either bygas or liquid introduced therein) causes membrane 25 to deflectdownward, thereby pinching off flow F passing through flow channel 30.Accordingly, by varying the pressure in channel 32, a linearly actuablevalving system is provided such that flow channel 30 can be opened orclosed by moving membrane 25 as desired. (For illustration purposesonly, channel 30 in FIG. 7G is shown in a “mostly closed” position,rather than a “fully closed” position).

Since such valves are actuated by moving the roof of the channelsthemselves (i.e.: moving membrane 25) valves and pumps produced by thistechnique have a truly zero dead volume, and switching valves made bythis technique have a dead volume approximately equal to the activevolume of the valve, for example about 100×100×10 μm=100 pL. Such deadvolumes and areas consumed by the moving membrane are approximately twoorders of magnitude smaller than known conventional microvalves. Smallerand larger valves and switching valves are contemplated in the presentinvention, and a non-exclusive list of ranges of dead volume includes 1aL to 1 uL, 100 aL to 100 nL, 1 fL to 10 nL, 100 fL to 1 nL, and 1 pL to100 pL.

The extremely small volumes capable of being delivered by pumps andvalves in accordance with the present invention represent a substantialadvantage. Specifically, the smallest known volumes of fluid capable ofbeing manually metered is around 0.1 μl. The smallest known volumescapable of being metered by automated systems is about ten-times larger(1 μl). Utilizing pumps and valves in accordance with the presentinvention, volumes of liquid of 10 nl or smaller can routinely bemetered and dispensed. The accurate metering of extremely small volumesof fluid enabled by the present invention would be extremely valuable ina large number of biological applications, including diagnostic testsand assays.

Equation 1 represents a highly simplified mathematical model ofdeflection of a rectangular, linear, elastic, isotropic plate of uniformthickness by an applied pressure:w=(BPb ⁴)/(Eh ³),  (1)where:

-   -   w=deflection of plate;    -   B=shape coefficient (dependent upon length vs. width and support        of edges of plate);    -   P=applied pressure;    -   b=plate width    -   E=Young's modulus; and    -   h=plate thickness.

Thus even in this extremely simplified expression, deflection of anelastomeric membrane in response to a pressure will be a function of:the length, width, and thickness of the membrane, the flexibility of themembrane (Young's modulus), and the applied actuation force. Becauseeach of these parameters will vary widely depending upon the actualdimensions and physical composition of a particular elastomeric devicein accordance with the present invention, a wide range of membranethicknesses and elasticity's, channel widths, and actuation forces arecontemplated by the present invention.

It should be understood that the formula just presented is only anapproximation, since in general the membrane does not have uniformthickness, the membrane thickness is not necessarily small compared tothe length and width, and the deflection is not necessarily smallcompared to length, width, or thickness of the membrane. Nevertheless,the equation serves as a useful guide for adjusting variable parametersto achieve a desired response of deflection versus applied force.

FIGS. 8A and 8B illustrate valve opening vs. applied pressure for a 100μm wide first flow channel 30 and a 50 μm wide second flow channel 32.The membrane of this device was formed by a layer of General ElectricSilicones RTV 615 having a thickness of approximately 30 μm and aYoung's modulus of approximately 750 kPa. FIGS. 21a and 21b show theextent of opening of the valve to be substantially linear over most ofthe range of applied pressures.

Air pressure was applied to actuate the membrane of the device through a10 cm long piece of plastic tubing having an outer diameter of 0.025″connected to a 25 mm piece of stainless steel hypodermic tubing with anouter diameter of 0.025″ and an inner diameter of 0.013″. This tubingwas placed into contact with the control channel by insertion into theelastomeric block in a direction normal to the control channel. Airpressure was applied to the hypodermic tubing from an external LHDAminiature solenoid valve manufactured by Lee Co.

While control of the flow of material through the device has so far beendescribed utilizing applied gas pressure, other fluids could be used.

For example, air is compressible, and thus experiences some finite delaybetween the time of application of pressure by the external solenoidvalve and the time that this pressure is experienced by the membrane. Inan alternative embodiment of the present invention, pressure could beapplied from an external source to a noncompressible fluid such as wateror hydraulic oils, resulting in a near-instantaneous transfer of appliedpressure to the membrane. However, if the displaced volume of the valveis large or the control channel is narrow, higher viscosity of a controlfluid may contribute to delay in actuation. The optimal medium fortransferring pressure will therefore depend upon the particularapplication and device configuration, and both gaseous and liquid mediaare contemplated by the invention.

While external applied pressure as described above has been applied by apump/tank system through a pressure regulator and external miniaturevalve, other methods of applying external pressure are also contemplatedin the present invention, including gas tanks, compressors, pistonsystems, and columns of liquid. Also contemplated is the use ofnaturally occurring pressure sources such as may be found inside livingorganisms, such as blood pressure, gastric pressure, the pressurepresent in the cerebrospinal fluid, pressure present in the intra-ocularspace, and the pressure exerted by muscles during normal flexure. Othermethods of regulating external pressure are also contemplated, such asminiature valves, pumps, macroscopic peristaltic pumps, pinch valves,and other types of fluid regulating equipment such as is known in theart.

As can be seen, the response of valves in accordance with embodiments ofthe present invention have been experimentally shown to be almostperfectly linear over a large portion of its range of travel, withminimal hysteresis. Accordingly, the present valves are ideally suitedfor microfluidic metering and fluid control. The linearity of the valveresponse demonstrates that the individual valves are well modeled asHooke's Law springs. Furthermore, high pressures in the flow channel(i.e.: back pressure) can be countered simply by increasing theactuation pressure. Experimentally, the present inventors have achievedvalve closure at back pressures of 70 kPa, but higher pressures are alsocontemplated. The following is a nonexclusive list of pressure rangesencompassed by the present invention: 10 Pa-25 MPa; 100 Pa-10 Mpa, 1kPa-1 MPa, 1 kPa-300 kPa, 5 kPa-200 kPa, and 15 kPa-100 kPa.

While valves and pumps do not require linear actuation to open andclose, linear response does allow valves to more easily be used asmetering devices. In one embodiment of the invention, the opening of thevalve is used to control flow rate by being partially actuated to aknown degree of closure. Linear valve actuation makes it easier todetermine the amount of actuation force required to close the valve to adesired degree of closure. Another benefit of linear actuation is thatthe force required for valve actuation may be easily determined from thepressure in the flow channel. If actuation is linear, increased pressurein the flow channel may be countered by adding the same pressure (forceper unit area) to the actuated portion of the valve.

Linearity of a valve depends on the structure, composition, and methodof actuation of the valve structure. Furthermore, whether linearity is adesirable characteristic in a valve depends on the application.Therefore, both linearly and non-linearly actuable valves arecontemplated in the present invention, and the pressure ranges overwhich a valve is linearly actuable will vary with the specificembodiment.

FIG. 9 illustrates time response (i.e.: closure of valve as a functionof time in response to a change in applied pressure) of a 100 μm×100μm×10 μm RTV microvalve with 10-cm-long air tubing connected from thechip to a pneumatic valve as described above.

Two periods of digital control signal, actual air pressure at the end ofthe tubing and valve opening are shown in FIG. 9. The pressure appliedon the control line is 100 kPa, which is substantially higher than the˜40 kPa required to close the valve. Thus, when closing, the valve ispushed closed with a pressure 60 kPa greater than required. Whenopening, however, the valve is driven back to its rest position only byits own spring force (≤40 kPa). Thus, τclose is expected to be smallerthan τopen. There is also a lag between the control signal and controlpressure response, due to the limitations of the miniature valve used tocontrol the pressure. Calling such lags t and the 1/e time constants τ,the values are: topen=3.63 ms, τopen=1.88 ms, τclose=2.15 ms,τclose=0.51 ms. If 3τ each are allowed for opening and closing, thevalve runs comfortably at 75 Hz when filled with aqueous solution.

If one used another actuation method which did not suffer from openingand closing lag, this valve would run at ˜375 Hz. Note also that thespring constant can be adjusted by changing the membrane thickness; thisallows optimization for either fast opening or fast closing. The springconstant could also be adjusted by changing the elasticity (Young'smodulus) of the membrane, as is possible by introducing dopant into themembrane or by utilizing a different elastomeric material to serve asthe membrane (described above in conjunction with FIGS. 7C-7H.)

When experimentally measuring the valve properties as illustrated inFIG. 9 the valve opening was measured by fluorescence. In theseexperiments, the flow channel was filled with a solution of fluoresceinisothiocyanate (FITC) in buffer (pH≥8) and the fluorescence of a squarearea occupying the center ˜⅓rd of the channel is monitored on anepi-fluorescence microscope with a photomultiplier tube with a 10 kHzbandwidth. The pressure was monitored with a Wheatstone-bridge pressuresensor (SenSym SCC15GD2) pressurized simultaneously with the controlline through nearly identical pneumatic connections.

6. Flow Channel Cross Sections

The flow channels of the present invention may optionally be designedwith different cross sectional sizes and shapes, offering differentadvantages, depending upon their desired application. For example, thecross sectional shape of the lower flow channel may have a curved uppersurface, either along its entire length or in the region disposed underan upper cross channel). Such a curved upper surface facilitates valvesealing, as follows.

Referring to FIG. 10, a cross sectional view (similar to that of FIG.7B) through flow channels 30 and 32 is shown. As can be seen, flowchannel 30 is rectangular in cross sectional shape. In an alternatepreferred aspect of the invention, as shown in FIG. 10, thecross-section of a flow channel 30 instead has an upper curved surface.

Referring first to FIG. 10, when flow channel 32 is pressurized, themembrane portion 25 of elastomeric block 24 separating flow channels 30and 32 will move downwardly to the successive positions shown by thedotted lines 25A, 25B, 25C, 25D, and 25E. As can be seen, incompletesealing may possibly result at the edges of flow channel 30 adjacentplanar substrate 14.

In the alternate preferred embodiment of FIG. 11, flow channel 30 a hasa curved upper wall 25A. When flow channel 32 is pressurized, membraneportion 25 will move downwardly to the successive positions shown bydotted lines 25A2, 25A3, 25A4 and 25A5, with edge portions of themembrane moving first into the flow channel, followed by top membraneportions. An advantage of having such a curved upper surface at membrane25A is that a more complete seal will be provided when flow channel 32is pressurized. Specifically, the upper wall of the flow channel 30 willprovide a continuous contacting edge against planar substrate 14,thereby avoiding the “island” of contact seen between wall 25 and thebottom of flow channel 30 in FIG. 10.

Another advantage of having a curved upper flow channel surface atmembrane 25A is that the membrane can more readily conform to the shapeand volume of the flow channel in response to actuation. Specifically,where a rectangular flow channel is employed, the entire perimeter (2×flow channel height, plus the flow channel width) must be forced intothe flow channel. However where an arched flow channel is used, asmaller perimeter of material (only the semi-circular arched portion)must be forced into the channel. In this manner, the membrane requiresless change in perimeter for actuation and is therefore more responsiveto an applied actuation force to block the flow channel

In an alternate aspect, (not illustrated), the bottom of flow channel 30is rounded such that its curved surface mates with the curved upper wall25A as seen in FIG. 20 described above.

In summary, the actual conformational change experienced by the membraneupon actuation will depend upon the configuration of the particularelastomeric structure. Specifically, the conformational change willdepend upon the length, width, and thickness profile of the membrane,its attachment to the remainder of the structure, and the height, width,and shape of the flow and control channels and the material propertiesof the elastomer used. The conformational change may also depend uponthe method of actuation, as actuation of the membrane in response to anapplied pressure will vary somewhat from actuation in response to amagnetic or electrostatic force.

Moreover, the desired conformational change in the membrane will alsovary depending upon the particular application for the elastomericstructure. In the simplest embodiments described above, the valve mayeither be open or closed, with metering to control the degree of closureof the valve. In other embodiments however, it may be desirable to alterthe shape of the membrane and/or the flow channel in order to achievemore complex flow regulation. For instance, the flow channel could beprovided with raised protrusions beneath the membrane portion, such thatupon actuation the membrane shuts off only a percentage of the flowthrough the flow channel, with the percentage of flow blockedinsensitive to the applied actuation force.

Many membrane thickness profiles and flow channel cross-sections arecontemplated by the present invention, including rectangular,trapezoidal, circular, ellipsoidal, parabolic, hyperbolic, andpolygonal, as well as sections of the above shapes. More complexcross-sectional shapes, such as the embodiment with protrusionsdiscussed immediately above or an embodiment having concavities in theflow channel, are also contemplated by the present invention.

In addition, while the invention is described primarily above inconjunction with an embodiment wherein the walls and ceiling of the flowchannel are formed from elastomer, and the floor of the channel isformed from an underlying substrate, the present invention is notlimited to this particular orientation. Walls and floors of channelscould also be formed in the underlying substrate, with only the ceilingof the flow channel constructed from elastomer. This elastomer flowchannel ceiling would project downward into the channel in response toan applied actuation force, thereby controlling the flow of materialthrough the flow channel. In general, monolithic elastomer structures asdescribed elsewhere in the instant application are preferred formicrofluidic applications. However, it may be useful to employ channelsformed in the substrate where such an arrangement provides advantages.For instance, a substrate including optical waveguides could beconstructed so that the optical waveguides direct light specifically tothe side of a microfluidic channel.

7. Networked Systems

FIGS. 12A and 12B show a views of a single on/off valve, identical tothe systems set forth above, (for example in FIG. 7A). FIGS. 13A and 13Bshows a peristaltic pumping system comprised of a plurality of thesingle addressable on/off valves as seen in FIG. 12, but networkedtogether. FIG. 14 is a graph showing experimentally achieved pumpingrates vs. frequency for the peristaltic pumping system of FIG. 13. FIGS.15A and 15B show a schematic view of a plurality of flow channels whichare controllable by a single control line. This system is also comprisedof a plurality of the single addressable on/off valves of FIG. 12,multiplexed together, but in a different arrangement than that of FIG.12. FIG. 16 is a schematic illustration of a multiplexing system adaptedto permit fluid flow through selected channels, comprised of a pluralityof the single on/off valves of FIG. 12, joined or networked together.

Referring first to FIGS. 12A and 12B, a schematic of flow channels 30and 32 is shown. Flow channel 30 preferably has a fluid (or gas) flow Fpassing therethrough. Flow channel 32, (which crosses over flow channel30, as was already explained herein), is pressurized such that membrane25 separating the flow channels may be depressed into the path of flowchannel 30, shutting off the passage of flow F therethrough, as has beenexplained. As such, “flow channel” 32 can also be referred to as a“control line” which actuates a single valve in flow channel 30. InFIGS. 12 to 15, a plurality of such addressable valves are joined ornetworked together in various arrangements to produce pumps, capable ofperistaltic pumping, and other fluidic logic applications.

Referring to FIGS. 13A and 13B, a system for peristaltic pumping isprovided, as follows. A flow channel 30 has a plurality of generallyparallel flow channels (i.e.: control lines) 32A, 32B and 32C passingthereover. By pressurizing control line 32A, flow F through flow channel30 is shut off under membrane 25A at the intersection of control line32A and flow channel 30. Similarly, (but not shown), by pressurizingcontrol line 32B, flow F through flow channel 30 is shut off undermembrane 25B at the intersection of control line 32B and flow channel30, etc.

Each of control lines 32A, 32B, and 32C is separately addressable.Therefore, peristalsis may be actuated by the pattern of actuating 32Aand 32C together, followed by 32A, followed by 32A and 32B together,followed by 32B, followed by 32B and C together, etc. This correspondsto a successive “101, 100, 110, 010, 011, 001” pattern, where “0”indicates “valve open” and “1” indicates “valve closed.” Thisperistaltic pattern is also known as a 120° pattern (referring to thephase angle of actuation between three valves). Other peristalticpatterns are equally possible, including 60° and 90° patterns.

In experiments performed by the inventors, a pumping rate of 2.35 nL/swas measured by measuring the distance traveled by a column of water inthin (0.5 mm i.d.) tubing; with 100×100×10 μm valves under an actuationpressure of 40 kPa. The pumping rate increased with actuation frequencyuntil approximately 75 Hz, and then was nearly constant until above 200Hz. The valves and pumps are also quite durable and the elastomermembrane, control channels, or bond have never been observed to fail. Inexperiments performed by the inventors, none of the valves in theperistaltic pump described herein show any sign of wear or fatigue aftermore than 4 million actuations. In addition to their durability, theyare also gentle. A solution of E. Coli pumped through a channel andtested for viability showed a 94% survival rate.

FIG. 14 is a graph showing experimentally achieved pumping rates vs.frequency for the peristaltic pumping system of FIG. 13.

FIGS. 15A and 15B illustrates another way of assembling a plurality ofthe addressable valves of FIG. 12. Specifically, a plurality of parallelflow channels 30A, 30B, and 30C are provided. Flow channel (i.e.:control line) 32 passes thereover across flow channels 30A, 30B, and30C. Pressurization of control line 32 simultaneously shuts off flowsF1, F2 and F3 by depressing membranes 25A, 25B, and 25C located at theintersections of control line 32 and flow channels 30A, 30B, and 30C.

FIG. 16 is a schematic illustration of a multiplexing system adapted toselectively permit fluid to flow through selected channels, as follows.The downward deflection of membranes separating the respective flowchannels from a control line passing thereabove (for example, membranes25A, 25B, and 25C in FIGS. 15A and 15B) depends strongly upon themembrane dimensions. Accordingly, by varying the widths of flow channelcontrol line 32 in FIGS. 15A and 15B, it is possible to have a controlline pass over multiple flow channels, yet only actuate (i.e.: seal)desired flow channels. FIG. 16 illustrates a schematic of such a system,as follows.

A plurality of parallel flow channels 30A, 30B, 30C, 30D, 30E and 30Fare positioned under a plurality of parallel control lines 32A, 32B,32C, 32D, 32E and 32F. Control channels 32A, 32B, 32C, 32D, 32E and 32Fare adapted to shut off fluid flows F1, F2, F3, F4, F5 and F6 passingthrough parallel flow channels 30A, 30B, 30C, 30D, 30E and 30F using anyof the valving systems described above, with the following modification.

Each of control lines 32A, 32B, 32C, 32D, 32E and 32F have both wide andnarrow portions. For example, control line 32A is wide in locationsdisposed over flow channels 30A, 30C and 30E. Similarly, control line32B is wide in locations disposed over flow channels 30B, 30D and 30F,and control line 32C is wide in locations disposed over flow channels30A, 30B, 30E and 30F.

At the locations where the respective control line is wide, itspressurization will cause the membrane (25) separating the flow channeland the control line to depress significantly into the flow channel,thereby blocking the flow passage therethrough. Conversely, in thelocations where the respective control line is narrow, membrane (25)will also be narrow. Accordingly, the same degree of pressurization willnot result in membrane (25) becoming depressed into the flow channel(30). Therefore, fluid passage thereunder will not be blocked.

For example, when control line 32A is pressurized, it will block flowsF1, F3 and F5 in flow channels 30A, 30C and 30E. Similarly, when controlline 32C is pressurized, it will block flows F1, F2, F5 and F6 in flowchannels 30A, 30B, 30E and 30F. As can be appreciated, more than onecontrol line can be actuated at the same time. For example, controllines 32A and 32C can be pressurized simultaneously to block all fluidflow except F4 (with 32A blocking F1, F3 and F5; and 32C blocking F1,F2, F5 and F6).

By selectively pressurizing different control lines (32) both togetherand in various sequences, a great degree of fluid flow control can beachieved. Moreover, by extending the present system to more than sixparallel flow channels (30) and more than four parallel control lines(32), and by varying the positioning of the wide and narrow regions ofthe control lines, very complex fluid flow control systems may befabricated. A property of such systems is that it is possible to turn onany one flow channel out of n flow channels with only 2(log 2n) controllines.

8. Switchable Flow Arrays

In yet another novel embodiment, fluid passage can be selectivelydirected to flow in either of two perpendicular directions. An exampleof such a “switchable flow array” system is provided in FIGS. 17A to17D. FIG. 17A shows a bottom view of a first layer of elastomer 90, (orany other suitable substrate), having a bottom surface with a pattern ofrecesses forming a flow channel grid defined by an array of solid posts92, each having flow channels passing therearound.

In preferred aspects, an additional layer of elastomer is bound to thetop surface of layer 90 such that fluid flow can be selectively directedto move either in direction F1, or perpendicular direction F2. FIG. 17Bis a bottom view of the bottom surface of the second layer of elastomer95 showing recesses formed in the shape of alternating “vertical”control lines 96 and “horizontal” control lines 94. “Vertical” controllines 96 have the same width therealong, whereas “horizontal” controllines 94 have alternating wide and narrow portions, as shown.

Elastomeric layer 95 is positioned over top of elastomeric layer 90 suchthat “vertical” control lines 96 are positioned over posts 92 as shownin FIG. 17C and “horizontal” control lines 94 are positioned with theirwide portions between posts 92, as shown in FIG. 17D.

As can be seen in FIG. 17C, when “vertical” control lines 96 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 98 will be deflected downwardly over the array of flow channelssuch that flow in only able to pass in flow direction F2 (i.e.:vertically), as shown.

As can be seen in FIG. 17D, when “horizontal” control lines 94 arepressurized, the membrane of the integrated structure formed by theelastomeric layer initially positioned between layers 90 and 95 inregions 99 will be deflected downwardly over the array of flow channels,(but only in the regions where they are widest), such that flow in onlyable to pass in flow direction F1 (i.e.: horizontally), as shown.

The design illustrated in FIGS. 17A-D allows a switchable flow array tobe constructed from only two elastomeric layers, with no vertical viaspassing between control lines in different elastomeric layers required.If all vertical flow control lines 94 are connected, they may bepressurized from one input. The same is true for all horizontal flowcontrol lines 96.

9. Cell Pen

In yet a further application of the present invention, an elastomericstructure can be utilized to manipulate organisms or other biologicalmaterial. FIGS. 18A-18D show plan views of one embodiment of a cell penstructure in accordance with the present invention.

Cell pen array 4400 features an array of orthogonally-oriented flowchannels 4402, with an enlarged “pen” structure 4404 at the intersectionof alternating flow channels. Valve 4406 is positioned at the entranceand exit of each pen structure 4404. Peristaltic pump structures 4408are positioned on each horizontal flow channel and on the vertical flowchannels lacking a cell pen structure.

Cell pen array 4400 of FIG. 18A has been loaded with cells A-H that havebeen previously sorted. FIGS. 18B-18C show the accessing and removal ofindividually stored cell C by 1) opening valves 4406 on either side ofadjacent pens 4404 a and 4404 b, 2) pumping horizontal flow channel 4402a to displace cells C and G, and then 3) pumping vertical flow channel4402 b to remove cell C. FIG. 18D shows that second cell G is moved backinto its prior position in cell pen array 4400 by reversing thedirection of liquid flow through horizontal flow channel 4402 a. Thecell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access.

While the embodiment shown and described above in connection with FIGS.18A-18D utilizes linked valve pairs on opposite sides of the flowchannel intersections, this is not required by the present invention.Other configurations, including linking of adjacent valves of anintersection, or independent actuation of each valve surrounding anintersection, are possible to provide the desired flow characteristics.With the independent valve actuation approach however, it should berecognized that separate control structures would be utilized for eachvalve, complicating device layout.

10. Cell Cage

The cell pen array 4404 described above is capable of storing materialswithin a selected, addressable position for ready access. However,living organisms such as cells may require a continuous intake of foodsand expulsion of wastes in order to remain viable. Accordingly, FIGS.19A and 19B show plan and cross-sectional views (along line 45B-45B′)respectively, of one embodiment of a cell cage structure in accordancewith the present invention.

Cell cage 4500 is formed as an enlarged portion 4500 a of a flow channel4501 in an elastomeric block 4503 in contact with substrate 4505. Cellcage 4500 is similar to an individual cell pen as described above inFIGS. 18A-18D, except that ends 4500 b and 4500 c of cell cage 4500 donot completely enclose interior region 4500 a. Rather, ends 4500 a and4500 b of cage 4500 are formed by a plurality of retractable pillars4502.

Specifically, control channel 4504 overlies pillars 4502. When thepressure in control channel 4504 is reduced, elastomeric pillars 4502are drawn upward into control channel 4504, thereby opening end 4500 bof cell cage 4500 and permitting a cell to enter. Upon elevation ofpressure in control channel 4504, pillars 4502 relax downward againstsubstrate 4505 and prevent a cell from exiting cage 4500.

Elastomeric pillars 4502 are of a sufficient size and number to preventmovement of a cell out of cage 4500, but also include gaps 4508 whichallow the flow of nutrients into cage interior 4500 a in order tosustain cell(s) stored therein. Pillars 4502 on opposite end 4500 c aresimilarly configured beneath second control channel 4506 to permitopening of the cage and removal of the cell as desired.

11. Cross-Channel Injector

The cross-flow channel architecture illustrated shown in FIGS. 18A-18Dcan be used to perform functions other than the cell pen just described.For example, the cross-flow channel architecture can be utilized inmixing applications.

This is shown in FIGS. 20A-D, which illustrate a plan view of mixingsteps performed by a microfabricated structures in accordance anotherembodiment of the present invention. Specifically, portion 7400 of amicrofabricated mixing structure comprises first flow channel 7402orthogonal to and intersecting with second flow channel 7404. Controlchannels 7406 overlie flow channels 7402 and 7404 and form valve pairs7408 a-b and 7408 c-d that surround each intersection 7412.

As shown in FIG. 20A, valve pair 7408 c-d is initially opened whilevalve pair 7408 a-b is closed, and fluid sample 7410 is flowed tointersection 7412 through flow channel 7404. Valve pair 7408 a-b is thenactuated, trapping fluid sample 7410 at intersection 7412.

Next, as shown in FIG. 20B, valve pairs 7408 c-d are closed and 7408 a-bare opened, such that fluid sample 7410 is injected from intersection7412 into flow channel 7402 bearing a cross-flow of fluid. The processshown in FIGS. 20A-B can be repeated to accurately dispense any numberof fluid samples down cross-flow channel 7402.

While the embodiment of a process-channel flow injector structure shownin FIGS. 20A-B feature channels intersecting at a single junction, thisis not required by the present invention. Thus FIG. 20C shows asimplified plan view of another embodiment of an injection structure inaccordance with the present invention, wherein junction 7450 betweenintersecting flow channels 7452 is extended to provide additional volumecapacity. FIG. 20D shows a simplified plan view of yet anotherembodiment of an injection structure in accordance with the presentinvention, wherein elongated junction 7460 between intersecting flowchannels 7462 includes branches 7464 to provide still more injectionvolume capacity.

12. Rotary Mixing Structure

Microfluidic control and flow channels in accordance with embodiments ofthe present invention may be oriented to rotary pump design whichcirculates fluid through a closed circuit flow channel. As used hereinthe term “closed circuit” has the meaning known in the art and refers toconfigurations that are circular and variations thereof such asellipsoids and ovals, as well as flow circuit paths having corners asare created by triangular, rectangular, or more complex shapes.

As illustrated in FIG. 21, a layer with flow channels 2100 has aplurality of sample inputs 2102, a mixing T-junction 2104, a centralcirculation loop 2106 (i.e., the substantially circular flow channel),and an output channel 2108. The overlay of control channels with a flowchannel can form a microvalve. This is so because the control and flowchannels are separated by a thin elastomeric membrane that can bedeflected into the flow channel or retracted therefrom.

The substantially circular central loop and the control channels thatintersect with it form the central part of the rotary pump. The pump(s)which cause solution to be flowed through the substantially circularflow channel consist of a set of at least three control channels 2110a-c that are adjacent to one another and which intersect thesubstantially circular branch flow channel 2106 (i.e., the centralloop).

When a series of on/off actuation sequences, such a 001, 011, 010, 110,100, 101, are applied to the control channels, the fluid in the centralloop can be peristaltically pumped in a chosen direction, eitherclockwise or counterclockwise. The peristaltic pumping action resultsfrom the sequential deflection of the membranes separating the controlchannels and flow channel into or out of the flow channel.

In general, the higher the actuation frequency, the faster the fluidrotates through the central loop. However, a point of saturation mayeventually be reached at which increased frequency does not result infaster fluid flow. This is primarily due to limitations in the rate atwhich the membrane can return to an unactuated position.

While the system shown in FIG. 21 shows each pump including threecontrol channels, a different number of control channels can beutilized, for example, a single serpentine control channel havingmultiple cross-over points could be used.

A variety of different auxiliary flow channels which are in fluidcommunication with the central loop can be utilized to introduce andwithdrawn sample and reactant solutions from the central loop.Similarly, one or more exit or outlet flow channels in fluidcommunication with the central loop can be utilized to remove solutionfrom the central loop. For example, control valves can be utilized atthe inlet(s) and the outlet(s) to prevent solution flow into or out fromthe central loop.

Flow channel sizes and shapes can vary. With certain devices, thediameter of the channel tends to range from about 1 mm to 2 cm, althoughthe diameter can be considerably larger in certain devices (e.g., 4, 6,8, or 10 cm). Limits on how small the diameter of the circular flowchannel can be are primarily a function of the limits imposed by themultilayer soft lithography processes. Channel widths (either flow orcontrol) usually vary between 30 μm and 250 μm. However, channel widthin some devices is as narrow as 1 um. Channels of larger widths can alsobe utilized, but generally require some type of structural supportwithin the flow channel. Channel height generally varies between 5 and50 μm. In flow channels having a width of 100 μm or less, the channelheight may be 1 μm or smaller. The flow channel is typically rounded toallow for complete blockage of the channel once the membrane isdeflected into the channel. In some devices, the channels have shapessuch as octagons or hexagons. In certain devices, the flow channels arerounded and 100 μm wide and 10 μm high and control channels are 100 μmwide and 10 μm high. One system that has been utilized in certainstudies has utilized a central loop having a diameter of 2 cm, a flowchannel width of 100 μm and a depth of 10 μm.

While the channels typically have the foregoing sizes and shapes, itshould be recognized that the devices provided herein are not limited tothese particular sizes and shapes. For example, branches present in aclosed circuit flow channel may serve to control the dispersion andhence mixing of materials flowed therein.

13. Microfluidic Large-Scale Integration

The previous section has described monolithic microvalves that aresubstantially leakproof and scalable, and has also described methods forfabricating these microvalves. For the relatively simple assemblies ofmicrofluidic valves previously described, each fluid flow channel may becontrolled by its own individual valve control channel. However, such anon-integrated control strategy cannot be practicably implemented formore complex assemblies comprising thousands or even tens of thousandsof individually addressable valves. Accordingly, a variety of techniquesmay be applied alone or in combination to allow for the fabrication oflarge scale integrated microfluidic devices having individuallyaddressable valves.

Techniques useful for implementing large scale integrated microfluidicstructures in accordance with embodiments of the present invention arediscussed in detail in U.S. nonprovisional patent application Ser. No.10/670,997, now U.S. Pat. No. 7,143,785. One technique allowing for thefabrication of large scale integrated microfluidic devices is the use ofmultiplexor structures.

The use of multiplexor structures has previously been described inconnection with a single set of control lines overlying a single set offlow channels. FIG. 22A shows a simplified plan view illustrating amicrofluidic binary tree multiplexor operational diagram. Flow channels1900 defined in a lower elastomer layer contain the fluid of interest,while control channels 1902 defined in an overlying elastomer layerrepresent control lines containing an actuation fluid such as air orwater. Valves 1904 are defined by the membranes formed at theintersection of the wider portion 1902 a of a control channel 1902 witha flow channel 1900. The actuation pressure is chosen so that only thewide membranes are fully deflected into the flow channel 1900.Specifically, the multiplexor structure is based on the sharp increasein pressure required to actuate a valve as the ratio of control channelwidth:flow channel width is decreased.

The multiplexor structure shown in FIG. 22A is in the form of a binarytree of valves where each stage selects one out of two total groups offlow channels. In the multiplexor embodiment shown in FIG. 22A, eachcombination of open/closed valves in the multiplexor selects for asingle channel, so that n flow channels can be addressed with only 2 log₂n control channels.

By using multiplexed valve systems, the power of the binary systembecomes evident: only about 20 control channels are required tospecifically address 1024 flow channels. This allows a large number ofelastomeric microvalves to perform complex fluidic manipulations withinthese devices, while the interface between the device and the externalenvironment is simple and robust.

FIG. 22B shows a simplified plan view of an alternative embodiment of amultiplexor structure in accordance with the present invention.Multiplexor structure 1950 comprises control channels 1952 formed in anelastomer layer overlying flow channels 1954 of an underlying elastomerlayer. Operating under the same physical principles of the multiplexorof FIG. 22A, multiplexor 1950 comprises a tertiary tree of valves, whereeach stage comprises three bits (“a trit”) and selects one out of threetotal groups of flow channels. Each combination of open/closed valves inthe multiplexor 1950 selects for a single channel, so that n flowchannels can be addressed with only 3 log ₃n control channels.

The general microfluidic flow architecture of either of the basicmultiplexor devices shown in FIGS. 22A-B may be generically representedin the simplified cross-sectional view of FIG. 22C, wherein secondelastomer layer E2 defining control channel network C overlies firstelastomer layer E1 defining flow channel network F.

The base 3 multiplexor of FIG. 22B is the most efficient design that maybe used to address large numbers of ‘flow” channels. This is because thex log_(x) n valve is minimized where e is used for the base of the log.As fractions are not used for the base of an actual multiplexor, themost efficient multiplexor structure is achieved where the value of x=3,the integer closest to e (˜2.71828).

To highlight this point, TABLE 2 compares the efficiency of the base 2multiplexor with the base 3 multiplexor.

TABLE 2 Number of Flow Lines Controlled by Control Lines EnhancedEfficiency Number of Base 2 Base 3 of Base 3 Multiplexor Control LinesMultiplexor Multiplexor Structure 6 8 9 +1 9 23 27 +4 12 64 81 +17 15181 243 +62 18 512 729 +217

While the above description has focused upon various multiplexorstructures utilizing stages having the same base number, this is notrequired by the present invention. Alternative embodiments ofmultiplexor structures in accordance with the present invention maycomprise stages of unlike base numbers. For example, a two-stagemultiplexor consisting of a bit stage and a trit stage represents themost efficient way of addressing six flow channels. The order of thestages is arbitrary, and will always result in the same number of flowlines being controlled. The use of multiplexor structures comprisingdifferent binary and tertiary stages allows the efficient addressing ofany number of “flow” channels that are the product of the numbers 2 and3.

A multiplexor may conceivably use any base number. For example, five mayalso be used as the base number, if necessary. However, efficiency inutilization of control lines diminishes as the number of control linesmoves away from the value of e. This is shown in FIG. 23, which plotsthe number of control lines versus the number of flow lines beingcontrolled, for multiplexor structures having different base numbers.

Another technique allowing for the fabrication of large scale integrated(LSI) microfluidic devices is the use of multiple layers of controllines. FIGS. 24-24B illustrate this approach. FIG. 24 shows a plan viewof one embodiment of a microfluidic device having a first control linecontrolled by a second control line. FIG. 24A shows a cross-sectionalview of the microfluidic device of FIG. 24, taken along line A-A′. FIG.24B shows a cross-sectional view of the microfluidic device of FIG. 24,taken along line B-B′.

Microfluidic structure 2100 comprises two flow channels 2102 a-b formedin lowermost elastomer layer 2104. First control channel network 2106including first inlet 2106 a in fluid communication with first andsecond branches 2106 b and 2106 c, is formed in a second elastomer layer2108 overlying first elastomer layer 2104. First branch 2106 b of firstcontrol channel network 2106 includes widened portion 2110 overlyingfirst flow channel 2102 a to define first valve 2112. Second branch 2106c of first control channel network 2106 includes widened portion 2114overlying second flow channel 2102 b to define second valve 2116.

Second control channel network 2118 comprising third control channel2118 a is formed in third elastomer layer 2120 overlying secondelastomer layer 2108. Third control channel 2118 a includes widenedportion 2118 b overlying first branch 2106 b of first control channelnetwork 2106 to form valve 2122.

The microfluidic device illustrated in FIGS. 24-24B may be operated asfollows. A fluid that is to be manipulated is present in flow channels2102 a and 2102 b. Application of a pressure to the first controlchannel network 2106 causes the membranes of valves 2112 and 2116 todeflect downward into their respective flow channels 2102 a and 2102 b,thereby valving flow through the flow channels.

Application of a pressure to second control channel network 2118 causesthe membrane of valve 2122 to deflect downward into underlying firstbranch 2106 c only of first control channel network 2106. This fixes thevalve 2112 in its deflected state, in turn allowing the pressure withinthe first control channel network 2106 to be varied without affectingthe state of valve 2112.

The general architecture of the microfluidic device depicted in FIGS.24-24B is summarized in the simplified cross-sectional view of FIG. 25.Specifically, elastomeric device 2200 comprises lowest elastomer layerE1 defining flow channel network F, underlying second elastomer layer E2defining first control channel network C1. First control channel networkC1 in turn underlies second control channel network C2 that is definedwithin third elastomer layer E3.

While the embodiment of the microfluidic device of FIGS. 24-24B isdescribed as being fabricated from three separate elastomer layers, thisis not required by the present invention. Large scale integratedmicrofluidic structures in accordance with embodiments of the presentinvention featuring multiplexed control lines may be fabricatedutilizing only two elastomer layers. This approach is shown andillustrated in connection with FIGS. 26-26B.

FIG. 26 shows a simplified plan view of a microfabricated elastomerdevice including first and second flow channels 2300 a and 2300 b, andfirst branched control channel network 2302 overlying flow channels 2300a and 2300 b to define valves 2304 and 2306 respectively. FIG. 26A showsa cross-sectional view of the microfabricated elastomer device of FIG.26, taken along line A-A′, with flow channel 2300 a defined in lowerelastomer layer 2306, and first control channel 2302 defined in upperelastomer layer 2310.

Lower elastomer layer 2308 further comprises a second control channelnetwork 2312 running underneath first control channel 2302 to definevalve 2314. Accordingly,

FIG. 26B shows a cross-sectional view of the microfabricated elastomerdevice of FIG. 26, taken along line B-B′. While present in the same(lower) elastomer layer 2308, flow channel network 2300 and secondcontrol channel network 2312 are separate and do not intersect oneanother.

As represented in the simplified cross-sectional view of FIG. 27,separate flow channel network F and control channel network C2 may thusbe present on a single (lower) elastomer layer E1 that is overlaid byanother elastomer layer E2 defining only a control channel network C1.

The microfluidic device illustrated in FIGS. 26-26B may be operated asfollows. A fluid that is to be manipulated is present in flow channels2300 a and 2300 b. Application of a pressure to the first controlchannel network 2302 causes the membranes of valves 2304 to deflectdownward into their respective flow channels 2300 a and 2300 b, therebyvalving flow through the flow channels.

Application of a pressure to second control channel network 2312 causesthe membrane of valve 2314 to deflect upward into the overlying branchof first control channel network 2302. This fixes the valve 2314 in itsdeflected state, in turn allowing the pressure within the first controlnetwork 2302 to be varied without affecting the state of valve 2314. Incontrast with the embodiment shown in FIG. 24, the microfluidic deviceof FIGS. 26-26B features a valve that operates by deflecting upward intoan adjacent control channel in response to an elevated pressure.

FIG. 27A shows an optical micrograph of a microfluidic comparator chip3000 microfabricated with large scale integration technology which isanalogous to an array of 256 comparators. Specifically, a second devicecontaining 2056 microvalves was designed which is capable of performingmore complex fluidic manipulations. The various inputs have been loadedwith colored food dyes to visualize the channels and sub-elements of thefluidic logic. FIG. 27B shows a simplified schematic plan view of oneportion of the chip of FIG. 27A.

Comparator chip 3000 is formed from a pair of parallel, serpentine flowchannels 3002 and 3004 having inlets 3002 a and 3004 a respectively, andhaving outlets 3002 b and 3004 b respectively, that are intersected atvarious points by branched horizontal rows of flow channels 3006.Portions of the horizontal flow channels located between the serpentineflow channels define mixing locations 3010.

A first barrier control line 3012 overlying the center of the connectingchannels is actuable to create adjacent chambers, and is deactivable toallow the contents of the adjacent chambers to mix. A second barriercontrol line 3014 doubles back over either end of the adjacent chambersto isolate them from the rest of the horizontal flow channels.

One end 3006 a of the connecting horizontal flow channel 3006 is influid communication with pressure source 3016, and the other end 3006 bof the connecting horizontal flow channel 3006 is in fluid communicationwith a sample collection output 3018 through multiplexor 3020.

FIGS. 27C-H show simplified enlarged plan views of operation of onemixing element of the structure of FIGS. 27A-B. FIG. 27C shows themixing element prior to loading, with the mixer barrier control line andwrap-around barrier control line unpressurized. FIG. 27D showspressurization of the wrap-around barrier control line and barrier mixerline to activate isolation valves and separation valve to defineadjacent chambers 3050 and 3052. FIG. 27E shows loading of the chamberswith a first component and a second component by flowing these materialsdown the respective flow channels. FIG. 27F shows pressurization of thevertical compartmentalization control line 3025 and the isolation todefine the adjacent chambers.

FIG. 27G shows depressurization of the mixing barrier control channel todeactivate the separation barrier valve, thereby allowing the differentcomponents present in the adjacent chambers to mix freely.

FIG. 27H shows the deactivation of barrier the isolation control line,causing deactivation of the isolation valves, followed by application ofpressure to the control line and deactivation of the multiplexor toallow the combined mixture to be recovered.

In the case of the device shown in FIGS. 27A-H, two different reagentscan be separately loaded, mixed pair wise, and selectively recovered,making it possible to perform distinct assays in 256 sub-nanoliterreaction chambers and then recover a particularly interesting reagent.The microchannel layout consists of four central columns in the flowlayer consisting of 64 chambers per column, with each chamber containing˜750 pL of liquid after compartmentalization and mixing. Liquid isloaded into these columns through two separate inputs under low externalpressure (˜20 kPa), filling up the array in a serpentine fashion.Barrier valves on the control layer function to isolate the samplefluids from each other and from channel networks on the flow layer usedto recover the contents of each individual chamber. These networksfunction under the control of a multiplexor and several other controlvalves.

The storage array and comparator microfluidic devices shown in FIGS.27A-H was fabricated with multilayer soft lithography techniques usingtwo distinct layers. The “control” layer, which harbors all channelsrequired to actuate the valves, is situated on top of the “flow” layer,which contains the network of channels being controlled. A valve iscreated where a control channel crosses a flow channel. The resultingthin membrane in the junction between the two channels can be deflectedby hydraulic or pneumatic actuation. All biological assays and fluidmanipulations are performed on the “flow” layer.

Master molds for the microfluidic channels were made by spin-coatingpositive photoresist (Shipley SJR 5740) on silicon 9 μm high andpatterning them with high resolution (3386 dpi) transparency masks. Thechannels on the photoresist molds were rounded at 120° C. for 20 minutesto create a geometry that allows full valve closure.

The devices were fabricated by bonding together two layers of two-partcure silicone (Dow Corning Sylgard 184) cast from the photoresist molds.The bottom layer of the device, containing the “flow” channels, isspin-coated with 20:1 part A:B Sylgard at 2500 rpm for 1 minute. Theresulting silicone layer is ˜30 μm thick. The top layer of the device,containing the “control” channels, is cast as a thick layer (˜0.5 cmthick) using 5:1 part A:B Sylgard using a separate mold. The two layersare initially cured for 30 minutes at 80° C.

Control channel interconnect holes are then punched through the thicklayer (released from the mold), after which it is sealed, channel sidedown, on the thin layer, aligning the respective channel networks.Bonding between the assembled layers is accomplished by curing thedevices for an additional 45-60 minutes at 80° C. The resultingmultilayer devices are cut to size and mounted on RCA cleaned No. 1, 25mm square glass coverslips, or onto coverslips spin coated with 5:1 partA:B Sylgard at 5000 rpm and cured at 80° C. for 30 minutes, followed byincubation at 80° C. overnight.

Simultaneous addressing of multiple non-contiguous flow channels isaccomplished by fabricating control channels of varying width whilekeeping the dimension of the flow channel fixed (100 μm wide and 9 μmhigh). The pneumatic pressure in the control channels required to closethe flow channels scales with the width of the control channel, makingit simple to actuate 100 μm×100 μm valves at relatively low pressures(˜40 kPa) without closing off the 50 μm×100 μm crossover regions, whichhave a higher actuation threshold.

Introduction of fluid into these devices is accomplished through steelpins inserted into holes punched through the silicone. Unlikemicromachined devices made out of hard materials with a high Young'smodulus, silicone is soft and forms a tight seal around the input pins,readily accepting pressures of up to 300 kPa without leakage.Computer-controlled external solenoid valves allow actuation ofmultiplexors, which in turn allow complex addressing of a large numberof microvalves.

II. Characterization and Analysis of Bacteria and Viruses

The microbial ecology of the human gut and other previously inaccessibleenvironments may be characterized and analyzed utilizing microfluidicchips to make genomic DNA (gDNA) libraries from individual bacteria in ahighly parallel fashion. The gDNA libraries can be analyzed using highthroughput screening or hybridization assays. The ability ofmicrofluidic structures to generate reagents from individual bacteriaenables the application of functional genomics to address previouslyinsoluble problems.

FIG. 28 shows a simplified flow chart of a generic process 6000 forcharacterization and analysis in accordance with one embodiment of thepresent invention. In step 6002, a sample containing bacteria and/orviruses is collected from the environment of interest. In step 6004, thebacteria or virus of interest is substantially physically isolated fromother components of the collected sample.

In step 6006, the isolated bacteria or virus is lysed to provide accessto the nucleic acid contents thereof. In step 6008, the nucleic acidexposed by cell/virus lysis is purified.

In optimal steps 6010 and 6012, the purified nucleic acid may becharacterized and amplified, respectively. Although FIG. 28 shows thestep of nucleic acid characterization occurring before amplification,this order of steps is not required by the present invention. Inaccordance with alternative embodiments, the purified nucleic acid maybe amplified prior to characterization.

In still other optional steps, nucleic acid that has been purified inaccordance with embodiments of the present invention, may be expressed.Specifically, step 6014 shows the ligation of the purified nucleic acidinto a plasmid or vector. Step 6016 shows the transformation of a hostcell with the ligated nucleic acid. Step 6018 shows expression of thenucleic acid by the transformed cell.

As described above, one or all of the steps of process 6000 may beperformed utilizing microfluidic architectures in accordance withembodiments of the present invention. The following sections summarizeparticular functions for isolation and characterizing bacteria andviruses, illustrating particular examples of microfluidic structuressuitable for performing these functions.

1. Bacterial/Viral Isolation

FIG. 29A shows a plan view of one embodiment of a microfluidicarchitecture in accordance with the present invention. Microfluidic chip6100 comprises first flow channel 6102 leading successively into first,second, and third gated cross-flow injector structures 6104, 6106, and6108. Gated cross-flow injectors 6104, 6106, and 6108 are defined by theintersection of first flow channel 6102 and second flow channel network6110 comprising branches 6110 a, 6110 b, and 6110 c. Three parallelisolations are available with different sample volumes of 1.6 nl, 1.0nl, and 0.4 nl, for cross-flow injection structures 6104, 6106, and6108, respectively. FIG. 29B shows an enlarged view of cross-flowinjection structure 6104.

Horizontal flow control line 6112 overlaps flow channels 6102 and 6110at the inlets and outlets to cross-flow injector structures 6104, 6106,and 6108, thereby controlling the flow of liquid into and out of thesestructures.

Prior to operation of the embodiment of the fluidic device shown in FIG.29, a sample has been obtained from the environment of interest. Samplesobtained from the environment may have bacterial/viral concentrations of1×10⁸⁻⁹ entity/ml or even higher. Such a concentration corresponds toabout 1 bacterial or viral entity per picoliter (pL), with onepicoliter=1000 nanoliters. If necessary, the concentration of the samplemay be diluted to ensure that the maximum number of desired bacterial orviral entities are captured within a microfluidic chamber having aparticular volume. Dilution may also be helpful in order to obtaindesired sample properties such as viscosity or pH.

In the initial, isolation step, the sample is flowed through inlet 6102a of first flow channel 6102. Because of the previous dilution step, alimited number of viruses/cells from the sample are present in each ofgated cross-flow injector structures 6104, 6106, and 6108. Horizontalcontrol line 6112 is then actuated to trap the diluted samples withincross-flow injector structures 6104, 6106, and 6108.

In one experiment, a culture of eGFP-expressing E. coli was verified toreach a final cell concentration of 5.3±3.5×10⁸ cells/mL. It was thendiluted 1:10 in nuclease-free water and loaded into the chip. Theaverage number of bacteria in the 0.4 nL, 1.0 nL, and 1.6 nLcompartments were 27, 61, and 139, respectively, in fair agreement withthe expected results of 21, 53, and 85.

While some dilution of the sample for isolation purposes may take placeoff-chip, a certain degree of sample dilution may also be accomplishedutilizing the microfluidic structure of FIG. 29 itself. Specifically,located immediately upstream of cross-flow injector structures 6104,6106, and 6108, are second cross-flow injector structures 6120, 6122,and 6124, respectively. Second cross-flow injector structures 6120, 122,and 6124 are defined by the intersection of third flow channel 6126 withbranches 6110 a-c of second flow channel network 6110.

A buffer may be located through inlet 6126 a of third flow channel 6126into cross-flow injector structures 6120, 6122, and 6124. The horizontalvalve 6130 separating the pairs of cross-flow injection structures6104/6120, 6106/6122, and 6106/6124, may then be deactivated to allowthe contents of these adjacent injection structures to mix, therebyfurther diluting the sample.

While the above description has focused upon isolation of a particularbacterium or virus through physical containment of a diluted sample in amicrofluidic chamber, other approaches could initially isolate bacteriaor viruses utilizing a sorting approach. One example of a microfluidicstructure useful for such sorting application is described by Fu et al,S.R. Anal. Chem. Vol. 74, pp. 2451-2457 (2002), hereby incorporated byreference for all purposes.

2. Lysis and Purification of Viral/Bacterial Nucleic Acid

In order to purify and recover nucleic acid from a particular cell orvirus, the cell or virus must first be lysed to expose the contentsthereof. Returning to FIG. 29A, inlet 6110 d to second flow channelnetwork 6110 is in fluid communication with three inlet flow channels.

Inlet 6110 a of second flow channel network 6110 is in fluidcommunication with lysis chemical inlet 6132 through valve 6133. Inlet6110 a of second flow channel network 6110 is in fluid communicationwith wash inlet 6134 through valve 6135. Inlet 6110 a of second flowchannel network 6110 is in fluid communication with elution inlet 6136through valve 6137.

During the lysis stage of operation of the microfluidic structure ofFIG. 29, lysis chemistry is flowed into flow channel network 6110. Thehorizontal valve isolating the cross-flow injection structures areopened, and the entire sample, buffer, and lysis solution mixture isflowed into mixing structures 6170.

The combined sample/buffer/lysis solution mixture is flowed into mixer6170 by a process of dead-ended loading, as loop exit control line 6172remains actuated. Mixing control lines 6171 a-c are then actuated toflow the mixture around the circular flow channel to accomplish mixing.FIG. 29C shows a photograph of an enlarged view of the rotary mixingstructure having a volume of 5 nL.

In the experiment described above wherein an average number of bacteriaof 27, 61, and 139 were loaded into the 0.4 nL, 1.0 nL, and 1.6 nLcompartments, subsequent transfer of the loaded bacteria into the rotarymixer ranged from about 70% to 92%.

FIGS. 30A-B show photographs of a sample of E. coli bacteria prior to,and subsequent to, lysing, respectively. Lysing was accomplished bymixing with a lysis buffer comprising sodium hydroxide and urea. Afterlysis, the bacterial cells ceased active movement and fluorescent signalwas lost.

After bacteria or viruses have been isolated and lysed on the chip, thenext step is to purify the nucleic acids of interest. It is possible topurify genomic DNA from bacterial cells lysed on chip.

Returning again to FIG. 29A, once the combination of the sample, buffer,and lysis solution have been thoroughly mixed, wash inlet valve 6133exit valves 6140 of mixers 6171 are opened. Wash is flowed through flowchannel network 6110 and the contents thereof, including mixers 6170,the contents of which are in turn flowed through a third set ofcross-flow injection structures 6171, 6172, and 6174, respectively, intowaste 6180. FIG. 29D shows an enlarged view of one bead-centerscross-flow injection structure.

Cross-flow injection structures 6171, 6172 and 6174 have been loadedthrough fourth flow channel 6176 and reactuated valves from bead trapcontrol line 6186 with beads coated with a substance exhibiting affinityto particular nucleic acids. Examples of such beads are DYNABEADS®available from Dynal Biotech of Oslo Norway.

Deactuation of the bead horizontal control valves defined by controlline 6188 allows the fluid from the mixer to be exposed to the beads. Asthe lysed sample flows from the mixer through the third set ofcross-flow injection structures containing the coated beads, nucleicacids in the sample adhere to the coated beads, while the remainder ofthe sample and wash is discarded.

Next, an elution solvent is flowed from inlet 6136 through flow channelnetwork 6110 and through third cross-flow injection structures 6171 and6172 and 6174 containing the beads. As a result of the presence of theflowing elution solvent, nucleic acid formerly bound to the beads isreleased. This eluted nucleic acid is collected in one of collectionports 6180, 6182 and 6184, and may then be amplified and/or analyzed.

Based upon the manufacturer's specified minimum capacity for each bead,each column formed by the third cross-flow injection structures of FIG.29A was calculated to have a total capacity of 100 pg of DNA, enough topurify genomic DNA from some 20,000 bacteria.

Since elution liquid is flowed from single input 6136 to each of thethird cross-flow injection structures to simultaneously elute thepurified nucleic acid, it is important that each processor exhibit thesame fluidic resistance. Specifically, if the channel lengths vary, thenthe corresponding resistance will cause unequal fluid flow in theseparate processors, thus affecting the elution rates. Therefore, theoverall microfluidic channel lengths for each processor were identical,with the central flow channel including a serpentine portion to addcompensating flow channel length.

FIG. 32A shows the results of electrophoresis of genomic DNA purifiedfrom a chamber containing less than about 280 individual E. colibacterial cells. Bacterial genomic DNA was recovered out of the chip byusing elution buffer. The DNA in the elution buffer was amplified withppdD primer sets by using conventional PCR method with 50 ul workingvolume.

The target nucleic acid purified was a 461 bp ppdD (prepilin peptidasedependent protein D precursor) gene from E. coli genome DNA. Genomic DNAcaptured on the surface of polystyrene magnetic beads were recovered byusing elution buffer and amplified by PCR reaction. The samples were runon 1.2% agarose gel in TBE (Tris-Borate-EDTA) buffer.

Gel lanes were as follows: L, R-100 bp DNA ladder; lanes 1, 4 and7—isolation of genomic DNA from 1.6 nL E. coli culture brothcorresponding cell number less than 1120; lanes 2, 5 and 8—isolation ofgenomic DNA from 1.0 nL of E. coli culture broth corresponding cellnumber less than 700; lanes 3, 5 and 9—isolation of genomic DNA from 0.4nL of E. coli culture broth with cell numbers less than 280. Lane 10; isa negative control.

FIG. 32B shows the results of electrophoresis analysis of genomic DNApurified from a chamber containing less than 28 individual E. colibacterial cells. Bacterial genomic DNA was recovered out of the chip byusing elution buffer.

The DNA in the elution buffer was amplified with ppdD gene primer setsby using a conventional PCR machine with 50 ul working volume. Again,the target purified nucleic acid is 461 bp ppdD (prepilin peptidasedependent protein D precursor) gene from E. coli genome DNA.

Gel lanes as follows: L, R-100 bp DNA ladder; lanes 1, 4 and 7—isolationof genomic DNA from 1.6 nL of 10-fold diluted E. coli culture brothcorresponding cell number less than 112; lanes 2, 5 and 8—isolation ofgenomic DNA from 1.0 nL of ten-fold diluted E. coli culture brothcorresponding cell number less than 70; lanes 3, 5 and 9—isolation ofgenomic DNA from 0.4 nL of ten-fold diluted E. coli culture brothcorresponding cell number less than 28. Lane 10 is a negative control.

FIGS. 31A-B show other electrophoresis results for nucleic acidspurified in accordance with embodiments of the present invention.Specifically, FIGS. 31A-B verify successful recovery of purified E. coligenomic DNA from the microfluidic architecture of FIG. 29A The elutedsamples were removed from the chip and amplified with PCR. The ampliconswere run on a 2.0% agarose gel in 0.5% TBE (Tris-Borate-EDTA) buffer.The target DNA is a 461 bp fragment of the E. coli ppdD (prepilinpeptidase dependent protein D precursor) gene.

FIG. 31A shows isolation of genomic DNA with undiluted E. coli culture,with the following gel lanes: M-PCR marker; lane 1—isolation of genomicDNA from 1.6 nL of culture with a corresponding cell number ofapproximately 1,120; lane 2—a 1.0 nL sample with a corresponding cellnumber of approximately 700; and lane 3—a 0.4 nL sample with cellnumbers of approximately 280. Lanes 1-3 of FIG. 31A show strong isolatedbands approximately 500 base pairs in length, revealing purification ofthe genomic DNA.

Lanes 4, 5, and 6 are negative controls for genomic DNA isolation inwhich purified water was used instead of cell culture sample and all theother conditions were the same as lanes 1, 2, and 3. No amplified signalwas present in any of these control cases.

FIG. 31B shows isolation of genomic DNA from a 1:10 dilution of cellculture. Lanes 1, 2, 3, lanes 4, 5, 6, and lanes 7, 8, 9 are the resultsfrom three different chips, respectively. Gel lanes as follows: M—PCRmarker; Lanes 1, 4, and 7—isolation of genomic DNA from an average of112 bacterial cells; Lanes 2, 5, and 8—isolation of genomic DNA from anaverage of 70 bacterial cells; Lanes 3, 6 and 9—isolation of genomic DNAfrom an average of 28 bacterial cells.

Since the amounts of purified DNA were too small to measure byconventional means, PCR amplification of the prelipin peptidasedependent protein (ppdD) gene was used for verification of successfulrecovery. The results of FIGS. 31A-B show that it is possible to reducethe number of cells needed for DNA isolation, and thereby increase thesensitivity of this process by 2,000-20,000 times over conventionalmethods.

The nucleic acid purification microfluidic architecture shown in FIGS.29A-D demonstrates the parallelization strategy employed. Process stepstake place along a roughly linear channel, allowing parallelization ofthe process in the orthogonal direction. This geometry allows a singlereagent fill line to dispense reagents simultaneously to all of theparallel processes.

The microfluidic architecture shown in FIGS. 29A-D was fabricated bymultilayer soft lithography. Mask designs were created with the CADprogram FluidArchitect (Fluidigm, South San Francisco, Calif.) andtransferred to high-resolution transparency masks (3,389 dpi). Thedimensions of the fluidic channels are 100 μm in width and 10 μm indepth, while the valve actuation channels are typically 200 μm in widthand 15 μm in depth. The chip included within 20×20 mm space, 13 fluidicaccess vias, 54 valves and 14 actuation vias that allow microfluidicflow and control.

Mask molds for the fluidic channels were made by spin-coating positivephotoresist (Shipley SJR 5740) on a silicon wafer with 2,000 rpm for 1min, followed by mask exposure and development. These mold channels arerounded at 135° C. for 15 min to create a geometry that allows fullvalve closure.

Another mold for the actuation layer is made by spinning photoresist ona separate wafer at 1,600 rpm for 1 min with a resulting height is 13μm, followed by mask exposure and development. The devices werefabricated by bonding together two layers of two-part cure silicone (GESilicone RTV615) cast from the photoresist molds.

The bottom layer of the device, containing the flow channels, is spincoated with 20:1 part A:B RTV615 at 2,400 rpm for 1 min and theresulting silicone layer is an 11 μm thick film. The top layer of thedevice, containing the actuation channels, is cast as a thick layer (5mm thick) with 5:1 part A:B RTV615 using a separate mold.

The two layers are initially cured for 30 min at 80° C. Actuationchannel interconnect holes are then punched through the thick layer witha 20 gauge luer stub, after which it is sealed, channel side down, onthe thin layer, after aligning the respective channel networks with anoptical microscope.

Bonding between the assembled layers is accomplished by curing theassembled devices for at 80° C. for more than 90 min, followed bypunching the fluidic channel interconnects. The resulting devices arecut to size and mounted on RCA cleaned cover slips (No. 1, 24 mm×50 mm),followed by incubation at 80° C. overnight to promote adhesion.

The chip containing the microfluidic architecture is mounted on aninverted microscope (Nikon Eclipse TE2000-S). Fluorescence excitationwas provided by a mercury lamp (100 W). A FITC filter set (Ex465-495, DM505, BA 515-555) was used and the image was recorded by using a PCcontrolled color digital camera (Sony DFW-V500).

Each actuation line on the chip was connected with a stainless steel pin(New England Small Tube, Litchfield, N.H.) and polyethylene tubing to anexternal solenoid valve controlled by a digital data I/O card (CCA,PPC1-DIO32HS; National Instruments, Austin, Tex.). Regulated externalpressure was provided to the normally closed port, allowing the controlchannel to be pressurized or vented to atmosphere by switching theminiature valve.

The fluidic vias for the introduction and collection of sample andbuffer were connected to an external pressure source throughpolypropylene tips (Multiplex tips, Sorenson BioScience, Inc., West SaltLake City, Utah). The typical pressure for driving liquid inside thechip is 0.5 to 2.0 psi (1 psi=6.89 kPa).

After the microfluidic chip was mounted on an optical microscope stageand the control channels are connected to external pneumatic controlsystems, microbeads (Dynabeads® DNA DIRECT™ universal, Dynal ASA, Oslo,Norway) with DNA binding capacity are loaded through the ‘bead in’ portin the upper right part of FIG. 29A. For this operation, valve 10 ‘loopexit control’ and valve 12 ‘bead trap control’ are closed. The beads arepositioned just before valve 12 by controlling the valves 6 and 12. Thepressure control for valve 12 is lower than the pressure for the othervalve controls, resulting in a partially closed valve that allows bufferto pass through while the beads accumulate into a column.

One μL of each of the buffer solutions (lysis buffer (Xtra Amp®, lysisbuffer series 1, Xtrana, Inc., Ventura, Calif.), wash buffer (Xtra Amp®,wash buffer series 1) and elution buffer (Tris-EDTA buffer, pH 7.8)) areloaded through the ‘lysis in’, ‘wash in’ and ‘elute in’ ports,respectively.

E. coli (BL21-2⁺) expressing eGFP were grown at 37° C. for 12 h inLuria-Bertani (LB) liquid medium containing ampicillin with 40 μg/ml ofconcentration. After the DNA purification and recovery from the chip, weattempted to amplify a gene, ppdD (prepilin peptidase dependent proteinD precursor, 461 bp), DNA contained in E. coli bacterial cells with aconventional PCR method.

Thermal cycling conditions were as follows: Initial denaturing: 2 min at95°; DNA denaturing: 30 sec at 95°; Primer annealing: 30 sec at 60°;dNTP polymerizing: 2 min and 30 sec at 72°; Repeat steps from denaturingto polymerizing 30 times; Final extension: 10 min at 72°.

The primer set for ppdD gene is 5′-GGTGGTTATTGGCATCATTGC-3′(SEQ ID NO:1)for forward and 5′-GTTATCCCAACCCGGTGTCA-3′ (SEQ ID NO:2) for reverse.The PCR cocktail is 5 μL of 10× reaction buffer, 1 μL of dNTP mix withthe final concentration of 10 mM, each 1 μL of forward and reverseprimers, 0.5 μL of Taq Polymerase 5 U/μL, and template and water to make50 μL of total reaction volume.

3. Lysis/Purification of Non-Bacterial Nucleic Acid

The above-referenced discussion relates to purification of nucleic acidsobtained from the lysis of bacterial cells. However, embodiments inaccordance with the present invention are not limited to purification ofthis type of nucleic acid. Other types of nucleic acid, including butnot limited to messenger RNA (mRNA) from eukaryotic cells, may beisolated and purified in accordance with embodiments of the presentinvention.

Isolation of mRNA, either to measure gene expression or to construct acDNA library, is a cornerstone of modern day molecular biology. Thereare numerous techniques for mRNA isolation, but nearly all requirehundreds to thousands of cells as a starting material. Yet there aremany situations where it would be useful to know the gene expressionprofile, or to develop a cDNA library, from a single cell.

Some primary cell types are very rare, like stem cells, and expansion inculture to acquire more cells changes the expression profile of thecell. Attempting to isolate a number of primary cells from an animal orpatient invariably results in a mixture of cell types because it is notpossible to precisely identify, and therefore isolate in a pure form,any single cell type. Furthermore, there has recently been a renewedinterest in epigenetic variations in gene expression between cells thatare nominally identical genotypes, to understand how these variationsmight play a role in development and other phenotypic differentiationprocesses.

There are well established methods for measuring expression of a fewgenes from a single cell, but these require a priori choice of a smallnumber of targets. This limitation has been somewhat alleviated byrecent progress in amplifying mRNA from a single cell for use in morehighly parallel microarrays. These methods allow parallel analysis of alarger number of targets, but the mRNA amplification process inevitablyintroduces some degree of distortion, and the microarrays themselvesrequire choice of a finite set of possible transcripts.

Construction of a cDNA library from a single cell has not yet beendemonstrated, but could provide a powerful tool to probe expressionwithout a priori assumptions about which genes are being probed. Thusfar, the minimum number of cells needed for cDNA library construction is1,000, with 10,000 being more usual, and the virtues of usingmicrofluidics to construct such a library have been recognized.

Accordingly, FIG. 37A shows an embodiment of a microfluidic architecture6400 in accordance with the present invention that is useful forisolation and purification of nucleic acid from other than bacteria orviruses. Lines 6402 represent the 100 μm wide fluidic channels, andlines 6404 are the 100 μm wide valve actuation channels. The fluidicports are named; the actuation ports are numbered 1 to 11.

Lysing buffer chamber 6408 comprises the channel space delineated byvalves 1, 2, 3 and 4. Cell chamber 6410 comprises the channel spacedelineated by valves 4, 5, 6 and 7. Beads chamber 6412 comprises thechannel space delineated by valves 7, 8, 9, and 10.

Nucleic acid purification chip 6400 comprises different functional unitsintegrated into a single structure. The processing architecture of thechip is horizontal, with reagents loaded vertically, a scheme which canbe generalized to implement a large class of sequential, batch processedreactions.

The first functional unit of architecture 6400 comprises chamber 6410that can be loaded with a variable number of cells. The precise numberof such loaded cells can be controlled by the concentration of cells inthe loaded solution. In this study, samples were diluted to trap between1 and 100 cells.

Adjoining the cell chamber 6410 is the lysis buffer chamber 6408, whichcan be loaded with a fixed amount of a solution of chaotropic salt.Valve 4 separating chambers 6408 and 6410 can be opened to allowdiffusion of the lysis buffer into the cells. The total volume in whichthe cells are lysed (between valves 1, 2, 3, 5, 6 and 7) is about 20 nL.

FIG. 37B is a photograph showing enlargement of the adjacent lysingbuffer and cell chambers of FIG. 37A. The actuation channels formingvalves 1 to 7 are filled with an aqueous solution of a food dye, orangeG. The scale bar is 200 microns.

The next functional unit of the chip is designed to create a packedcolumn of oligo dT derivatized paramagnetic beads (DynabeadsOligo(dT)25) through which the cell lysate can be flushed. In order totrap a precise amount of beads, a microfluidic valve whose opening couldbe controlled precisely by a pressure regulator, was used.

Valve 8 is left slightly open so that a regulated fluid flow can passthrough, but the large (2.8 gm) beads are trapped. All the other valvesare on off valves, controlled by individual pressure sources. Thosepressure sources are actuated by an NI DAQ card (National Instrument)and a graphic interface developed under Labview 6.0 (Fluidigm Corp). Atthe end of each run, beads are collected from the chip and assayed forthe presence of mRNA with benchtop RT PCR.

FIG. 37C is a photograph showing an enlargement of the adjacent bead andcell chambers of FIG. 37A. A column of 2.1 μm diameter paramagneticbeads covered with oligo dT is being built against a partially closedmicrofluidic valve 8. The scale bar is again 200 microns.

Specific operation of the microfluidic chip 6400 may be summarized asfollows. All the valve actuation lines were filled with fluid (aconcentrated aqueous solution of Orange G) in order to avoid bubbleformation in the fluidic channels. The membrane separating the fluidicchannel and the control line is very thin (less than 5 microns) andpermeable to air. Each control line is connected to its pressure source.

The working pressure at which all pressure sources were set was 2 psiabove the actuation pressure of the valves, thus allowing a 1 psipressure on the fluidic channels. The working pressure was differentfrom one chip to another due to small variations in the membranethickness, but typically between 8 and 15 psi. All the valves wereclosed before loading the reagents.

The reagents were delivered through polyethylene tubing, which wasconnected to the chip using 23 gauge stainless steel pins (New EnglandSmall Tubes, Litchfield, N.H.). The other end of the tubing wasconnected to a common pressure source for all the fluidic channel inputs(1 psi). The chip was loaded with 10 μl, of cell solution, 20 μL of alLysis buffer, and 5 μL of beads solution. The original Dynal beads wereresuspended in Dynal Lysis buffer and reconcentrated to 5×concentration.

The lysis buffer was first loaded on the chip by opening valves 1 and 2.Once the lysis buffer flowed through the Lysing Buffer Out channel,valve 1 was closed. The lysis buffer chamber was dead end filled bypushing the air into the gas permeable chip. Valve 2 was then closed.

Cells of the sample were prepared as follows. Freshly harvested NIH 3T3cells were used for each experiment. NIH 3T3 cells were grown to nearconfluency on 100 mm tissue culture plates. The cells were rinsed with1× PBS and then trypsinized. The trypsinized cells were resuspended in1× PBS in order to adjust the final cell concentration to between 106/mLand 107/mL.

The cells were loaded by opening valves 5 and 6. The cell suspensionflowed through the cell chamber, and valve 5 was closed. Valve 5 couldbe opened and closed repeatedly until a suitable number of cells weretrapped in the cell chamber. The number of cells trapped in the chambercould also be influenced by changing the cell suspension concentration.Typically, 1 to 100 cells could be trapped and lysed on the chip.

Valve 6 was kept open until the remaining air in the cell chamber waspushed out. Valve 6 was then closed.

The bead suspension was loaded by opening valves 9 and 10. Once the beadsuspension flowed through the beads chamber, valve 9 was closed and thechamber was dead end-filled by pushing the remaining air into the gaspermeable chip. Valves 8 and 11 were then opened and pressure on valve 8was slowly increased using the pressure regulator until the 2.1 pmdiameter beads began to stack up against the partially closed valve.Once the stack was sufficiently long to reach the Beads In channel,valve 10 was closed.

Valve 4 was opened, thereby allowing the lysis buffer to diffuse intothe cell chamber. As lysis buffer reached an individual cell, the cellwould immediately lyse, and then gradually disintegrate. FIG. 37D showsa photograph of an enlarged portion of the cell chamber after initialloading. FIG. 37E shows a photograph of an enlarged portion of the cellchamber after five minutes of diffusion of lysis chemical, as themembrane begins to disintegrate as the cell is lysed. FIG. 37F shows aphotograph of an enlarged portion of the cell chamber after ten minutesof diffusion, as the cell has completely disintegrated. The scale barsin FIGS. 37D-F are 100 microns.

The cell lysate was flushed out of the “cell chamber” by opening valves3, 4, 7 and 11. The lysate was allowed to flow through the stack ofbeads at a controlled speed (typically 100 μm/s) that could be adjustedby changing the pressure on the Air In channel. The poly A containingmRNA hybridized to the oligo dT on the beads and remained on the beadswhile the majority of the cell debris washed on out of the chip. Theair/fluid interface stops at the stack of beads due to surface tension.

Valve 3 was then closed, valve 2 was opened, and lysis buffer wasflushed through the beads to wash them free of as much cellular debrisas possible. Valve 2 was then closed

A short tubing was connected to the output port of the chip and placedin a 0.1 mL PCR tube. Valves 2 and 8 were opened and the beads wereflushed with lysis buffer into the PCR tube. A magnet was used to drawout any beads remaining in the outflow channel. The beads werecentrifuged, and then resuspended in 100 μL of fresh lysis buffer. RNaseInhibitor (1 μL) was added. The tube was vortexed and the mRNA waseither analyzed immediately, or the tube was stored frozen at 80° C.

In a typical experiment, several chips were used to obtain beadscarrying mRNA from one or more cells. Reverse transcription andamplification was carried out directly on the beads using the QiagenOneStep RT PCR kit, followed by gel electrophoresis of the products.Primers were used to identify two types of mRNA: the high abundance βactin transcript and the moderate abundance zinc finger ozf transcript.

FIG. 38 shows electrophoresis results for a series of experimentsperformed in order to demonstrate single cell sensitivity together withnegative controls. The RT PCR products were analyzed on a 2% agarose gelloaded with 5% of the reaction; the amplified gene is β actin.

Gel lanes are as follows: lane 1: 1 kb ladder; lane 2: PBS, no cells (onthe chip); lane 3: 1 cell (on the chip); lane 4: 9 cells (on the chip);lane 5: 200 uL supernatant from 1 day old cells +5 μL of beads (in atest tube); lane 6: 200 μL lysing buffer +5 μL of beads (in a testtube); lane 7: cells loaded on the chip but none trapped in the chamber(on the chip); lane 8: 200 μL, DI water +5 μL beads (in a test tube);lane 9: PCR reagents only.

FIG. 38 shows successful mRNA isolation from a single NIH 3T3 cell inlane 3. A positive control, supernatant from lysed NIH 3T3 cells, isshown in lane 5, and various negative controls are shown: All thenegative controls show no band in the (3 actin DNA region, while thepositive control demonstrates a strong band.

When the intensity of a band is normalized by the number of beads in thecolumn, a monotonically increasing relationship is found betweenintensity and cell number for the highly abundant O actin mRNA, down tothe single cell level. Specifically, a second series of experiments wasperformed in order to test the chips with variable numbers of cells andboth high and medium copy number transcripts.

The RT PCR products for both (β-actin and ozf were analyzed on a 2%agarose gel, whose bands were quantitated, normalized, and plotted inFIG. 39. Zero values indicate the absence of a detectable band in thegel; the experiment with 19 cells failed for both transcripts, possiblybecause of RNAase contamination or chip failure.

For high abundance actin mRNA (closed circles), detection is down to thesingle cell level. For the moderately abundant zinc finger ozf mRNA,signal could be detected from as little as two cells, but the typicalsensitivity is somewhere between 2 and 10 cells. Thus, the sensitivityof this first generation chip is established

Despite the fact that the RT PCR is not a linear amplification process,these results are semi quantitative in the sense that they areconsistent with the cell number. If the band intensities are notnormalized by the size of the column, then the results are not monotonicand do not obey any obvious functional relationship.

This result is interpreted as an indication that the kinetics of mRNAbinding to the beads were not fully equilibrated, and that thesensitivity of future devices can be improved by increasing theinteraction time of the lysate with the column.

An intriguing possible future application for microfluidic mRNApreparation is as a tool to generate subtractive libraries from singlecells (or more accurately, pairs of single cells). Such a procedurewould be valuable in eliminating commonly expressed transcripts whileenriching for differentially expressed transcripts, and is not feasiblefor small numbers of cells using conventional tools.

In a standard preparation, mRNA isolated from roughly 1×10⁶ cells isprocessed in a volume of order 10 μL. The same mRNA concentration wouldbe attained (assuming no adhering of mRNA to vessel walls) if the mRNAfrom 1 cell were contained in a 10 pL volume. While such volumes arelarger than those used in the present study, they are readily attainablewith current microfluidic technology, and valves with an order ofmagnitude smaller displacement volume have already been demonstrated.

One difference between the microfluidic architecture of FIG. 29Autilized for isolation and purification of bacterial nucleic acid, andthe microfluidic architecture of FIG. 37A utilized for isolation andpurification of non-bacterial nucleic acid, is the presence of an activemixing structure to enhance mixing of the lysing chemical and thesample. In the case of viruses or bacteria, such active mixing is usefulto rapidly disrupt the resilient viral sheath or cell wall structuresprotecting the virus or bacteria. However, entities other than bacteriaor viruses (such as mammalian cells), lack these protective structures,and may therefore experience lysis under less stringent conditions, forexample mere diffusion across a microfluidic free interface.

Another difference between the microfluidic architecture of FIG. 29Autilized for isolation and purification of bacterial nucleic acid, andthe microfluidic architecture of FIG. 37A utilized for isolation andcharacterization of non-bacterial nucleic acid, is the use of parallelflow structures controlled by common control lines. However, thistechnique can also be employed in the isolation and purificationnon-bacterial nucleic acid, as illustrated in connection with FIG. 40.

FIG. 40 shows a plan view of an alternative embodiment of a microfluidicarchitecture in accordance with the present invention. Microfluidicarchitecture 6500 allows eight simultaneous cDNA libraries to be createdby implementing the SMART cDNA library kit in chip format.

Left column 6502 of inputs can be connected to an elution buffermanifold. Top row 6504 contains control lines and reagent inputs; withthe ten reagent inputs comprising, in order, stem cells, lysis buffer,oligo dT beads, primers, reverse transcriptase, PCR reagents, proteinaseK, Swi I, plasmids, and competent bacteria. The top row flow and controlinlets would fan out to allow access to space for macroscopicconnections.

The parallel cDNA microfluidic architecture of FIG. 40 uses bothdiffusive and active rotary mixing, depending on the time sensitivity ofthe step. The eight separate libraries may be recovered from outputs6506.

Chip design 6500 combines cell lysis, mRNA purification, and a chipbased implementation of a commercial cDNA library kit. Temperature maybe controlled with a thermo electric stage mounted to the microscope,and reagents will be flushed into the vertical channels as needed, thuspreventing aging, denaturation or inappropriate temperatures duringearlier stages of the experiment.

Chip 6500 is designed to make a library of cDNA clones from a smallnumber (from 1 to 1,000) of cells. It uses the basic microfluidictechniques already described to isolate, lyse, and purify mRNA fromcells, which will then be cloned into competent E. coli cells using theCreator SMART cDNA Library Construction kit from Clontech. This kit canreadily be translated to chip format since it is a one tube, one stepreaction.

The bacteria will be recovered from the chip using the methods developedfor the cell sorter and MHTSC chips. Since the bacteria growexponentially, one can easily grow enough material to interrogate thelibrary using conventional genomic techniques: DNA microarrays and highthroughput EST sequencing being two examples. The chip may be validatedusing a cell line and comparing clone frequency to frequencies measuredby high throughput EST sequencing.

To evaluate the cDNA library constructed from a small number of cells onthe chip, the following steps may be followed. First, one large aliquotof cells (1×10⁸) will be used to isolate mRNA and check the integrity ona gel. A small aliquot (1 to 1000 cells) of the same cells will betrapped in the chip and the mRNA isolated and checked on a gel.

Conventional cDNA library construction will then be performed with theminimum of 1 ug mRNA; at the same time, a cDNA library will beconstructed on a chip. Initially, conventionally isolated mRNA will beused to verify that a cDNA library can be made on a chip. Later, thefirst stage chip (cells to mRNA) will be combined with the second part(mRNA to cDNA library).

Next, random clones (12) from each library will be characterized for theaverage insertion size. One hundred individual clones from each librarywill then be selected for sequence analysis and to establish a profilefor each cDNA library.

The redundant cDNA will be mixed and used to make a probe, andhybridization will be performed to the other 9900 colonies. The negativecolonies will next be sequenced. This procedure should greatly reducethe number of colonies that need to be sequenced, since the redundantcolonies will be eliminated.

Bioinformatic analysis will then be performed to compare the geneexpression profile between the conventional cDNA library constructionand the chip based cDNA library construction.

Library insert sizes will also be characterized. Obtaining full lengthtranscripts will not be crucial for the initial version of the chip, asbioinformatic tools for the analysis of the human genome will be at amature enough state that most gaps will be able to be filled in bycomputer. However, later versions of the chip for other applications canbe optimized for full length transcripts.

Isolation and purification of mRNA utilizing microfluidic techniques inaccordance with embodiments of the present invention may find use in anynumber of applications. One such application is the construction of cDNAlibraries. Another application is mRNA isolation for gene expressionanalysis, for example utilizing a microarray chip as is well known toone of ordinary skill in the art.

While the microfluidic architectures described above are employed in theligation and transformation of purified nucleic acids, the mixing andflow schemes may be generalized to encompass other usefulfunctionalities. For example, the mixing structures utilized in ligationor transformation can in turn be in fluid communication with othermixing structures used to prepare specific materials from chemical kits.Examples of materials prepared by such chemical kits include but are notlimited to specific plasmids for ligation, and specific enzymes utilizedin constructing cDNA libraries, as well known to one of ordinary skillin the art.

The above sections have described the isolation within a microfluidicchamber, of individual or subsets of elements of a heterogeneous sample.Embodiments in accordance with the present invention are not limited toisolation of any particular number of relevant biological entitieswithin a particular microfluidic chamber. Embodiments of microfluidicarchitectures and methods in accordance with the present invention couldbe designed to isolate 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1000, 5,000,10000, 50,000, 100,000 or an even greater number of biological entitieswithin a single microfluidic chamber.

4. Amplification of Nucleic Acid

Polymerase chain reaction (PCR) has become one of the most ubiquitoustools in molecular biology, and thus one of the most importantbiochemical reactions to be implemented in a microfluidic device is theability to amplify nucleic acids. Liu et al., “A Nanoliter Rotary Devicefor PCR”, Electrophoresis, Vol. 23, p. 1531 (2002), incorporated byreference herein for all purposes, have previously created amicrofluidic architecture for performing PCR.

FIG. 34 shows a schematic diagram of one embodiment of a microfluidicstructure 6300 in accordance with the present invention for performingPCR. Top layer 6302 includes control channels 6304 a-d for controllingthe pumps and valves. Middle layer 6304 defines the inlet 6305 a, rotary6305 b, and outlet 6305 c fluid flow channels. Bottom layer 6306includes integrated heater structures 6308 and electrical leads 6310 inelectrical communication therewith.

The loop in the fluid layer forms a rotary pump, by which the PCRreagents can be transported over regions of different temperatures. Thetemperatures are set by tungsten heaters evaporated onto glass, whichbecome the bottom substrate of the nanofluidic chip. The total volume ofthe PCR reaction was 12 nL.

FIG. 35 shows results of Taqman PCR assay performed in the rotary pumpchip. Fluorescence was measured in situ at various time points as thePCR reaction mixture was pumped through different temperature regions. Afragment of the β-actin gene was amplified from human male genomic DNA.The closed circles represent data from an experiment which contained thehuman template DNA. The open circles represent data from a negativecontrol experiment in which template DNA was withheld.

Amplification of purified nucleic acid samples in accordance withembodiments of the present invention is not limited to the specificmicrofluidic structure shown in FIG. 34. Alternative, more complexmicrofluidic geometries are possible. In accordance with one alternativeembodiment, a microfluidic architecture comprising a matrix of reactionchambers would allow n DNA samples to react with m primer pairs, therebyallowing m×n reactions to be performed simultaneously.

FIG. 36A shows a plan view of a matrix PCR architecture. FIG. 36B showsan enlarged view of one cell of the matrix PCR architecture of FIG. 36A.A complete description of the matrix PCR microfluidic architecture shownin FIGS. 36A-B is described in U.S. provisional patent application No.60/494,432, incorporated by reference herein for all purposes.

As shown in the schematic layout of FIGS. 36A-B, the device comprisesthree layers having distinct functions. A middle matrix flow channelstructure is sandwiched between a top layer comprising integratedhydraulic valves, and a lower layer comprising and pneumatic pumps. Inthe middle layer, the microfluidic flow channels are 106 μm wide and 1214 μm high. Each vertex of the matrix contains a rectangular-shapedchannel comprising a reactor, having a volume of ˜3 nL.

Each row of reactors is connected to a separate input port (˜625 μm indiameter) through which unique primers may be loaded. Each column cansimilarly load the reactors with different DNA templates. A single inputfor the addition of polymerase is connected to all the reactors in thematrix.

In the upper control layer, the valve system is designed to load eachreactor with the three separate reagents while preventing crosscontamination. In total, 2860 valves displayed horizontally orvertically are controlled by only 2 independent pressure supply throughholes. Furthermore, the large valves (B in the inset of FIG. 36, 270 μmwide) or the small valves (C, 96 μm wide) can be selectively actuatedbecause they have a different threshold of hydraulic pressure necessaryfor actuation.

Reagent loading is not blocked by the narrow control channels (42 μmwide) connecting the valve system because their tiny membrane does notdeflect at the actuation pressure used. The second, bottom control layercomprises a 20×20 array of rotary pumps in order to facilitate mixingthe reagents.

Active valves in the upper control layer facilitate the loading andisolation of reagents. With valves A actuated, primer sets are loadedalong each row of the matrix. Actuation of valves B and C isolates awell defined volume of primers in each reactor.

Valves A are then opened to allow for the loading of DNA templates downeach column, while polymerase is simultaneously introduced to allreactors from a single inlet. Valves A are once again actuated, definingthe desired volumes of polymerase and templates and isolating eachreactor.

The different membrane areas of valves B and C allow for the selectiveopening of valve C by reducing the actuation pressure from 260 to 110kPa, thus bringing all three components into fluidic contact within eachreactor. Peristaltic pumps in the bottom layer allow for the rapidrotary mixing of all reagents within the reactors.

A matrix PCR microfluidic device design as shown in FIGS. 36A-Brepresents an advance in addressing issues relating to interfacingnanofluidic chips to the macro world, because the DNA template andprimer samples are partitioned m and n fold, respectively on the chip.Furthermore, the polymerase, which is often the most expensive part ofthe sample, is partitioned by a factor of m×n. The design scales in astraightforward way, allowing fabrication of microfluidic devices wheren=20 and m=20, yielding 400 simultaneous reactions.

5. Ligation of Nucleic Acid

Once nucleic acid has been purified, it may be incorporated within acell and then expressed. The first step of such an expression process isto ligate the nucleic acid within a host vector such as a plasmid. FIG.33 shows a plan view of one embodiment of a microfluidic architecturesuitable for such ligation and expression of purified nucleic acid. Themicrofluidic structure 6200 of FIG. 33 comprises a first set of parallelmixing structures used for ligation of nucleic acid, and a second set ofparallel mixing structures used for transformation of cells with theligated, purified nucleic acid.

FIG. 33A shows an enlarged view of the region of the microfluidicarchitecture of FIG. 33 allowing ligation of purified nucleic acid.Control line 6205 bisects each of mixing structures 6201, 6202, 6203,creating fluidly isolated hemispheres. First hemisphere 6201 a of mixingstructure 6201 is in fluid communication with inlet flow channel 6207.First hemisphere 6202 a of second mixing structure 6202 is in fluidcommunication with first hemisphere 6201 a of first mixing structure6201 through linking flow channel 6209. First hemisphere 6203 a of thirdmixing structure 6203 is in fluid communication with first hemisphere6202 a of second mixing structure 6202 through linking flow channel6211. Actuation of control line 6205 allows the host vector/plasmid tobe flowed through inlet 6207 and links 6209 and 6211 into the firsthemispheres of all three mixing structures.

Second hemisphere 6201 b of mixing structure 6201 is in fluidcommunication with inlet flow channel 6213. Second hemisphere 6202 b ofsecond mixing structure 6202 is in fluid communication with firsthemisphere 6201 through linking flow channel 6215. Second hemisphere6203 b of third mixing structure 6203 is in fluid communication withsecond hemisphere 6202 b of second mixing structure 6202 through linkingflow channel 6217. Actuation of control line 6205 allows purifiednucleic acid mixed with ligation enzyme to be flowed through inlet 6213and branches 6215 and 6217 into the second hemispheres of all threemixing structures.

Each of second hemispheres 6201 b, 6202 b, and 6203 b of mixingstructures 6201, 6202, and 6203 are also in fluid communication withinlet flow channels 6221 through loading structure 6290. Specifically,while control line 6227 is actuated, fluid is flowed through flowchannel 6221. As a result of the closed state of the valves defined bycontrol line 6227, the fluid flowed down channel 6221 is diverted intoutilizing cross-connector flow channel 6229 and flow channels 6219 and6223. Because flow channels 6219 and 6223 are open-ended, loadingstructure 6290 may thus be rapidly filled with wash or other fluid,avoiding the delay that would otherwise be necessary to dead-end loadflow channel 6221.

Next, control line 6227 is deactuated, and control line 6225 actuated,such that application of pressure to flow channel 6221 results in thecontents of loading structure 6290 being flowed into second hemispheres6201 b, 6202 b, and 6203 b of mixing structures 6201, 6202, and 6203,respectively. In the manner just indicated, inlet 6221 may be utilizedto load the mixing structures with washing fluid and/or SOC media lateruseful during gene transformation.

Once the first and second hemispheres of mixing structures 6201, 6202,and 6203 have been loaded with the vector and ligation enzyme/purifiednucleic acid respectively, control channels 6231 a-c may be actuated ina systematic fashion to accomplish circulation within the mixingstructures. This circulation results in the enzyme causing ligation ofthe purified nucleic acid within the host vector/plasmid.

6. Transformation of Cells with Nucleic Acid

The microfluidic structure 6200 of FIG. 33 also comprises a second setof parallel mixing structures 6251, 6252, and 6253 used fortransformation of a cell with the ligated purified nucleic acid. FIG.33B shows an enlarged view of the region of the microfluidicarchitecture of FIG. 33 allowing cell transformation with the ligatedpurified nucleic acid.

The microfluidic architecture employed for cell transformation issimilar to that used for nucleic acid ligation. Specifically, the threeparallel mixing structures 6251, 6252, and 6253, are bisected by acontrol line 6255, creating fluidly isolated hemispheres. Firsthemisphere 6251 a of mixing structure 6251 is in fluid communicationwith inlet flow channel 6257. First hemisphere 6252 a of second mixingstructure 6252 is in fluid communication with first hemisphere 6251 a offirst mixing structure 6251 through linking flow channel 6259. Firsthemisphere 6253 a of third mixing structure 6253 is in fluidcommunication with first hemisphere 6252 a of second mixing structure6252 through linking flow channel 6261. Actuation of control line 6255allows the host cells to be flowed through inlet 6257 and links 6259 and6261 into the first hemispheres of all three parallel mixing structures6151-6153.

Second hemisphere 6251 b of mixing structure 6251 is in fluidcommunication with mixing structure 6201 through connecting flow channel6271. Second hemisphere 6252 b of mixing structure 6252 is in fluidcommunication with mixing structure 6202 through connecting flow channel6272. Second hemisphere 6253 b of mixing structure 6253 is in fluidcommunication with mixing structure 6203 through connecting flow channel6273. Control line 6275 overlies each of the connecting flow channels6171-6173 to define valves isolating the nucleic acid ligation region ofthe architecture from the cell transformation region of thearchitecture.

Once the first hemisphere of the mixing structures have been loaded withthe cells that are to be transformed, control line 6255 is deactuatedand the host vectors incorporating the ligated purified nucleic acidsare flowed from first mixing structures 6201, 6202, and 6203 into secondmixing structures 6251, 6252, and 6253, respectively. Control channels6177 a-c are actuated in a systematic fashion to accomplish circulationof the cells and host vectors within the mixing structures.

Upon application of a heat shock to the contents of the second mixingstructures, the cells incorporate the vector and the purified nucleicacid present therein. Such a heat shock may be applied across the entirefluidic chip, or alternatively may be applied only locally to the secondmixing structures utilizing small temperature control structuresfabricated for that purpose.

7. Culturing Transformed Cells

Once cells within the second mixing structures have been exposed to heatshock and thereby incorporated the host vector containing the purifiednucleic acid, they may be flowed from outlet flow channels 6179 a-c forculturing. In accordance with one embodiment, the transformed cellscould be removed from the chip for culturing. Alternatively, culturingof the transformed cells could take place directly on-chip in cell penor cage structures described above.

The cultured and transformed cells would be expected to express thepurified nucleic acid present in the incorporated vector. As describedabove in connection with an alternative approach to cell isolation, thecultured transformed cells could be sorted by an additional microfluidicstructure. This sorting could be based upon the physical characteristicsof the transformed cells resulting from their expression of the purifiednucleic acid.

8. Applications for Bacterial/Viral Nucleic Acid

Evolutionary Variation: Cells->gDNA->PCR->Recover DNA

One application for nucleic acid purification in accordance with thepresent invention is to study evolutionary variation in a restricted setof genes. Evolutionary relationships are currently deduced mainly on thebasis of either ribosomal RNA or whole genome comparisons, whenavailable. In accordance with embodiments of the present invention, amicrofluidic manipulation of a sample may allow proportional measurementof the distribution of mutations for selected genes in bacteria not ableto be cultured. The microfluidic architecture may isolate a singlebacterium from a sample, lyse that bacterium, and then purify thegenomic DNA from the bacterium. The microfluidic architecture may thenamplify a selected gene (or set of genes) using PCR. The amplifiedmaterial can be recovered from the chip, re-amplified, and sequenced.

gDNA Cloned Library: Cells->gDNA->Digestion->gDNA Library

In accordance with another embodiment in accordance with the presentinvention, a cloned library of genomic DNA may be created from a singleor small number of bacteria. Individual bacteria from a diluted samplecan be distributed into microfluidic chambers. The bacteria are thenlysed, the genomic DNA purified, digested, and then ligated into cloningvectors. These vectors will then be transformed into E. coli using heatshock and mixing as described in connection with FIG. 33.

A vector allowing an average insert size in the range 1-2 kbp may beused. The gDNA libraries can be recovered and analyzed usingconventional genomic methods. The gDNA library can be characterized byhybridization to a microarray, or representative sequencing. Cloningefficiency can be measured and evaluated.

Cloned BAC Library: Cells->gDNA->Digestion->BAC Library

In accordance with still other embodiments of the present invention,microfluidic architectures can be used to make a cloned bacterialartificial chromosome (BAC) library of genomic DNA from a single orsmall number of bacteria. These BAC libraries offer the advantage ofhaving much larger average insert sizes, on the order of ˜200 kbp.

Individual bacteria can be distributed into chambers on the chip. Theisolated bacteria can then be lysed, and the resulting genomic DNApurified, digested, and then ligated into cloning vectors. These vectorswill then be transformed into E. coli. Vectors and reagents to constructthe BACs can be supplied from known sources. The resulting BAC librariescan be recovered from the chip and analyzed using conventional genomicmethods. The recovered BAC library can be characterized by endsequencing. The cloning efficiency will be measured and evaluated.

Using a BAC library offers the advantage of preserving gene order,making reassembly easier. As in prior whole genome sequencing efforts,having both long and short insert libraries may be useful in achievingthe most complete possible genome assemblies.

Characterization of Microbial Diversity

For over a century termite gut contents had been cited as beingexcellent sources for the microscope-viewing of abundant andmorphologically diverse spirochetes. Spirochetes are anultrastructurally distinct and genetically coherent, phylum-levelassemblage of prokaryotes. Although prevalent in the guts of manytermites (sometimes accounting for as much as 50% of the direct counts),they also had the reputation for “not being cultivable”. However,Leadbetter et al. have amassed a 9 strain collection from the hindgutcontents of the California Dampwood Termite, Zootermopsis angusticollis,

The first 2 isolates obtained, ZAS-1 and ZAS-2, have been shown capableof H₂+CO₂ acetogenesis. In addition to being the first representativetermite gut isolates, these were the first in the phylum Spirochetesshown to be capable of chemolithotrophic metabolism. Additionally, thiswas the first demonstration of H₂+CO₂-acetogenic catabolism by anymicrobe not belonging to the phylum Firmicutes, class Clostridia.

Much can be learned about the function of the uncultivated microbialdiversity of the Zootermopsis hindgut. Leadbetter et al. obtained amolecular inventory of the bacterial community diversity of the hindgut.Different species of bacteria in a sample may have differences in theircell masses, surface areas, and rates of activity. Further complicatingmatters, different bacterial species may have anywhere from a single toover a dozen rRNA genes encoded on their genome. This makes it difficultto correlate gene abundance in a sample (as assessed by amplifying fromthe DNA) with that of the active biomass (which manifests itself astotal rRNA content) of those organisms encoding them.

In order to gain a better understanding of the active microbiota in thegut, cell volume, activity, and 16s gene copy number were “normalized”by deriving a molecular inventory from the rRNA itself. RNA purification(from total gut nucleic acids) and RT-PCR were used to construct a cDNAtemplate, from which an inventory was subsequently amplified and cloned.

This inventory focused on the recovery of bacterial sequences usingall-Bacteria primers, and thus does not yet include protozoal orarchaeal representatives. About 200 full 16s rRNA sequences were clonedand RFLP-sorted, identifying approximately 50 unique ribotypes. Over 20%of the clones recovered corresponded to an unknown microbe belonging toa deeply divergent bacterial lineage, “Leptospirillum and relatives”.The only studied microbes belonging to this phylum are 1) extremeacidophiles that live by oxidizing iron, 2) aerobes that live byoxidizing nitrite, or 3) thermophiles that respire sulfate to sulfide.

However, knowing the phylogeny of this microbe reveals little of itsphysiology or function. No niche in any animal gut for any microbe withany of such catabolism is known, and yet potentially a fifth of theactive microbial biomass in the termite hindgut, as deduced from theresults of the inventory, is from a microbe related to these with suchcatabolism. Because of the deduced abundance of these bacteria, theybecome excellent candidates for exploring the efficacy and impact ofcell sorting and further in depth analysis vis a vis nanofluidicdevices.

Microbial diversity in the human gut can also be characterized using thegDNA chips previously discussed. The following model can be used toestimate the quality of data expected from such experiments. Supposelibraries are created from a population of 1,000 individual bacteria,and that this population is made up of roughly 100 species, each ofwhich occurs 10 times. Further assume that each bacterial genome is 1mega base pair and that the average insert size is 1 kilo base pair. Ifthe gDNA chip were to clone with 100% efficiency, the resultinglibraries would have 10× coverage of each of the 100 bacterial species.Assuming a more realistic figure of 10% efficiency, then each specieswould have on average 1× coverage.

For any given species this coverage is spread over 10 differentlibraries, without any a priori knowledge which 10 out of the 1,000libraries this is. This leads to the natural question of, if when thelibraries are completely sequenced, whether there will be enough overlapbetween the libraries to link them together as belonging to the samespecies.

A simple calculation using the above figures shows that the probabilityof not being able to link any pair of libraries is roughly e⁻²⁰. Thus,after completely sequencing each library, any given library should beable to be associated with another originating from the same species.This allows complete deconvolving of the library data and reconstructionof the microbial diversity of the sample.

Thus in the hypothetical example given above, if the average sequencingread length was 500 base pairs, and all the bacterial genomes wereunknown, then 200,000 sequencing lanes per experiment would be required(i.e. per 1,000 bacteria). In practice, resequencing of known genomeswould be halted as soon as they are identified, which should be after asmall number of sequencing lanes.

An alternative set of experiments would explore incorporation of a cellsorting component into the front end of the gDNA chip just described.Antibody markers and FACS would be used to enrich the population forunknown bacteria, thereby allowing sorting of known species into thewaste channel, and collecting the unknown bacteria.

Many of the gut microbes have distinct and differing morphologies. Thiswill make it reasonably straight forward to identify and collect asignificant sample of the genetic diversity that underlies themorphological. BAC libraries of genomic DNA of collected individualswill be constructed. After this, clones from each BAC library will bescreened with primers designed to specifically amplify only the rRNAencoding gene of the not-yet-cultivated leptospirillum-relativeidentified earlier during our 16S rRNA molecular inventory.

Once identified, not only the BAC clones containing single genomic DNAfragments encoding the rDNA will be retrieved, but also libraries of theentire or near entire genome of the cells with that ribotype.

Microbial Ecology

Microbial ecology is an emerging field of study that explores therelationship between specific environments and the biological entitiesexisting therein. Investigation of the properties and genetic makeup ofsuch environmental biological entities can offer tremendous benefits, asexemplified by the discovery of antibiotic properties of the penicillinmold existing in the environment.

Fortuitously, penicillin proved amenable to laboratory cultivation, andits antibiotic properties were able to be investigated and harnessed.However, a relatively large percentage of bacterial cells and otherbiological entities from the environment have so far resisted laboratorycultivation. The continuing inability to reproduce these environmentalbiological entities under controlled conditions has so far precludedaccess to their genetic information and metabolic potential, limitinginvestigations of microbial ecology.

In an attempt to glean information regarding not-yet-cultivatedbiological entities present in specific ecological or clinicalenvironments, many researchers have turned to metagenomics. Inmetagenomics, samples comprising highly complex heterogeneous mixturesof biological entities are collected from an environment. Biologicalentities present within the samples are disrupted en masse, and theresulting nucleic acid and genetic information contained therein isprocessed first as a single unit—a “meta” genome corresponding to anentire environment rather than to a specific organism. Examples ofrecent metagenomic investigations include Lorenz et al., “Screening fornovel enzymes for biocatalytic processes: accessing the metagenome as aresource of novel functional sequence space”, J. Curr Opin Biotechnol.2002 December; 13(6):572-7, and Rondon et al., “Cloning the soilmetagenome: a strategy for accessing the genetic and functionaldiversity of uncultured microorganisms.”, Appl Environ Microbiol. 2000June; 66(6):2541-7, both of which are incorporated by reference hereinfor all purposes.

While metagenomics shows some promise for microbial ecologyinvestigations, it offers some serious drawbacks. One drawback ofmetagenomics approaches is dissolution of the link between the geneticinformation obtained (i.e. the nucleic acid), and the origin of thatinformation (i.e. the biological entity containing the nucleic acid).This link between information and source can be reconstructed from themorass of community biocomplexity only at great effort, if at all. Thususing such metagenome approaches, it has only been straightforward toidentify the genes as being from the same biological entity if theyco-locate on the same BAC genome fragment. The remainder of the genomiclibrary for that species is lost into a vast, unsortable BAC mixtureproduced by en masse disruption of all biological entities or componentspresent in the highly heterogeneous environmental or clinical sample.

By contrast, microfluidic techniques in accordance with embodiments ofthe present invention offer a promising alternative to metagenomicapproaches. Specifically, microfluidic methods and structures enablephysical isolation of individual or small numbers of biological entitieswithin a larger sample. Subsequent purification of nucleic acid presentwithin the individual or subset of biological entities can readily belinked to morphotypic, ribotypic, and genotypic information obtainedfrom the physically isolated biological entity or entities. Thiscontrasts with the metagenome approach to cloning, in which totalcommunity environmental DNA is restricted and cloned into a BAC library.A net sequencing of a microbial community is achieved utilizing bothmetagenomic and microfluidic techniques, but only in the latter approachis informational coherence of individual genomes maintained.

9. Applications for Non-Bacterial Nucleic Acid

The foregoing description has so far focused upon applications forpurification of nucleic acid from bacteria or viruses. However, thepresent invention is not limited to this particular application, and inaccordance with alternative embodiments, nucleic acids from mammalian orother non-bacterial cell types may be purified utilizing microfluidicapproaches, as discussed in detail in U.S. provisional patentapplication No. 60/494,388, filed Aug. 11, 2003 and incorporated byreference herein for all purposes.

Cells->mRNA->cDNA->Taqman PCR in Matrix

In accordance with one embodiment, a microfluidic chip can be designedto take a small number of cells (from 1 to 1,000), lyse them, purifymRNA, create cDNA, and detect the presence of specific transcripts usingTaqman PCR in a matrix geometry. Such a microfluidic architecture can beused to optimize the parameters of mRNA purification and cDNA synthesis.

Components for cell lysis, bead trapping, affinity purification, andTaqman PCR may be combined in order to purify and detect expression ofselected genes. The number of cells used to produce cDNA may besystematically varied, and the results compared from large numbers usedin conventional macroscopic techniques to chip-based results using 1,000cells, 100 cells, 10 cells, and 1 cell. Such a comparison would allowunderstanding of the effects of cell number stochasticity, anddetermination of the number of cells needed to make reliablemeasurements of medium and low copy number mRNA.

An advantage of such a microfluidic design is that it will allow directmeasurement of cDNA production efficiency without introducing furtherlosses in the cloning process. A well characterized cell line, such asNIH_MGC_53 or NIH_MGC_93, for which there is extensive EST andmicroarray data, could be used for these experiments. Using a cell lineinstead of tissue will control for possible bias in the results fromunidentified sub-populations in a heterogeneous sample. Validation maybe done by comparing chip results to both conventional results obtainedmanually, and to results listed in the NIH EST database (10,000 clonessequenced).

Cells->mRNA->cDNA->RNA->Microarray

In another application, a microfluidic chip could be designed to take asmall number of cells (from 1 to 1,000), lyse them, purify mRNA, createcDNA with a T7 promoter, and then use T7 RNA polymerase to make linearlyamplified, fluorescent RNA which can then be hybridized to a microarray.The microarray may be fabricated in situ onto the chip using methodssimilar to the surface derivatization previously demonstrated forsurface biotinylation.

Such a chip architecture would provide a more stringent test of cDNAquality than the first variation since the amplification stage islinear. Validation can be performed by comparing to DNA microarray dataobtained by conventional methods. Again, use of a cell line will controlfor unidentified subpopulations. Possible applications for such amicrofluidic architecture include tumor typing, clinical diagnosis,prediction of treatment outcome, and developmental biology.

Cells->mRNA->cDNA->Differential Cloned Library

In still another application, a microfluidic chip may be designed toconstruct a library of cDNA clones from a small number (from 1 to 1,000)of cells. Such a chip may use the methods of the original cDNA chip inorder to construct the library.

In this application, however, two cell populations will be processed inparallel, and before cloning the libraries will be normalized againsteach other. Such a chip architecture can be validated using two celllines for which conventional techniques can be used. The resultinglibrary elements can be sequenced.

Hematopoietic Stem Cell Studies

The pluripotent hematopoietic stem cells (PHSC) are a rare population ofcells that reside in the bone marrow and that maintain the dynamics ofthe blood system. Following intrinsic characteristics and environmentalinfluences, these cells will move into one of the following pathways:self renewal as primitive stem cells, maturation into differentiatedhematopoietic cells, or apoptosis. In adult bone marrow, a balance amongthese three pathways is maintained in order to provide normalhematopoiesis.

Based on the limited dilution competitive repopulation assay, thefrequency of PHSC in bone marrow is approximately 1 in 100,000 nucleatedcells. Isolation of PHSC is a necessary step in studying their function.

The most reliable method in the field to isolate PHSC is to use FACSsorting using a variety of cell membrane surface proteins as markers.Purified mouse PHSC has been obtained by developing a 5 color FACSsorting system using a 3 laser MoFlo Cytometer with Summit data analysissoftware (other investigators use 3 color sorting based on a threesurface protein expression profile; several groups can do 4 colorsorting).

With this 5 color system, bone marrow cells were labeled with 5different antibodies (lineage markers cocktail, anti Sca 1, anti c kit,antiCD38 and anti CD34) conjugated with different fluorochromes. Themost primitive hematopoietic stem cells are characterized by the surfaceexpression profile: LinSca 1+kit+CD38+CD34 (abbreviated as +++− cells).These cells (called long term repopulating cells, LTRC) were shown toprovide long term repopulation of the blood system in lethallyirradiated mice.

FIGS. 41A-B show flow cytometric analysis of the surface markerexpression profile of murine bone marrow cells. Lineage-positive cellswere removed by CS column before flow cytometry. FIG. 41A showsexpression of Sca 1 and c kit on the cell surface was gated as shown;Lin−Sca+kit+ cells were gated as shown in the box A.

FIG. 41B shows expression of CD38 and CD34 on the cell surface of theLin−Sca+kit+ cells was used to separate the subpopulation shown in box Aof the left figure into the following 4 subsets: Sca+kit+CD38+CD34,Sca+kit+CD38+CD34+, Sca+kit+CD38−CD34+, and Sca+kit+CD38−CD34−. Cells ineach population were sorted and collected for analysis in a repopulationassay. Based on FIGS. 41A-B, the frequency in bone marrow nucleatedcells was shown to be 2 in 100,000 nucleated cells.

Further analysis indicated that the physiological pathway appears to be:+++− cells differentiate next into ++++ cells (Lin Sca 1+kit+CD38+CD34+)and then into ++−+ cells (Lin Sca 1+kit+CD38−CD34+). Both ++++ and ++−+cells belong to the category of short term repopulating cells (STRC),since they can only repopulate a lethally irradiated mouse for a numberof weeks, not for the normal lifetime of the animal. However, both LTRCand STRC are needed to rescue a lethally irradiated recipienteffectively.

From the above observations, the hypothesis of PHSC regulation ismodeled as following. FIG. 42 illustrates a proposed regulatory networkfor murine pluripotent hematopoietic stem cells. As illustrated, signals(positive and/or negative) between these three bone marrow subsetsregulates the balance of cells within the stem cell compartment in thebone marrow, thereby controlling hematopoiesis. Preliminary evidence hasbeen obtained to support the partial regulatory network shown in FIG.42.

Understanding the regulatory mechanisms that control hematopoiesis inthe human should make it possible to manipulate the system in vivo. Suchcontrol would have a broad range of clinical applications, including incancer therapy, treatment of blood diseases, genetic disorders, as wellas stem cell gene therapy.

One of the approaches for gaining an insight into the regulation ofhematopoiesis is to identify the genes that are specifically expressedat each stage of cell maturation and then to determine their function inthe hematopoietic pathway. Genes that were specifically expressed in+++− cells, while not being expressed in ++++ or ++−+ cells, i.e., wereidentified to determine those genes that specifically provide an LTRCphenotype.

Specifically, using differential display PCR (DD PCR), 184 genetranscripts were identified from a total of 1395 DD PCR fragments, assignificantly elevated or uniquely expressed in +++− cells, comparedwith ++++ and ++−+ cells. These 184 gene fragments were confirmed byreverse northern blots, subcloned, and sequenced.

Three known data bases, Genbank, EST (expression sequenced tag), andstem cell data base from Princeton university (developed by IhorLemishka), were screened. Seventy-two of the genes (39%) exhibitedhomology to genes with known function. Fifteen genes (8%) exhibitedhomology to sequences in the EST data base.

Ninety-seven genes (53%) appear to be novel, i.e., they do not havehomology to any sequence in the three examined databases. The Celeradatabase can be screened for these “unknown” genes. To obtain the fulllength version of these “unknown” genes, a commercial 17 day mouseembryo cDNA library was first screened. However, only 10.1% of the genesgave a positive signal in a PCR reaction.

The embryo cDNA library was derived from a mixture of cell types.Consequently, low expression transcripts from any single cell type wouldbe buried in the background.

Accordingly, two cDNA libraries were constructed using whole mouse bonemarrow as a starting point: one library was from “low density cells” andthe second from “lineage negative” cells. Rather than needingapproximately 1×10⁸ cells as required for commercial cDNA librarypreparations, by scaling down each step, reasonable libraries with1×10⁴⁻⁵ cells have been obtained.

The low density cell and lineage negative cell cDNA libraries wereconstructed with the technology developed by Clontech and Gibco BRL,respectively. A tracer of radioactivity (32P dCTP) was used in analiquot from each library construction to measure 1″ and 2″ a strandsynthesis efficiency.

Twelve random clones were picked, and DNA was prepared followed byrestriction enzyme digestion. The average size of the insert was 1.5 kb(with an insert size range of 0.8 kb to 3.1 kb) for the low density celllibrary and an average of 2.2 kb for the lineagenegative cell library,respectively. However, a total of 109 and 108 cells were required,respectively.

Comparing the positive PCR reactions using the “unknown” gene fragmentsfor each of the three cDNA libraries, the observed frequency ofpositives was highest in the lineage negative cell cDNA library: 30%,lineage negative library; 18%, low density library; 10%, commercial 17day embryo library. Nonetheless, +++− cells are still a relatively rarepopulation even in the lineage negative library (50 in 100,000 cells).Seventy of the genes remain missing.

The appropriate resolution would be to construct a cDNA library from+++− cells. However, obtaining 1×10⁵ +++− cells would be a verydifficult task. Approximately 200 +++− cells can be obtained from onemouse. Obtaining 1×10⁵ +++− cells would require, therefore, harvestingbone marrow cells from 500 mice, a logistically impossible task for anacademic laboratory. Furthermore, even 5 color sorting does not providea pure population of cells.

Accordingly, production of a cDNA library on a chip from 1 or only a fewcells in accordance with embodiments of microfluidic approaches inaccordance with the present invention, would address both problems.Specifically, use of microfluidics would involve only a limited numberof cells, and would result in relatively pure material due to thereduction of a mixture of cells in any population of primary cells.

While the above description has focused upon the use of microfluidicarchitectures for isolation and purification of nucleic acids fromviruses, bacteria, and multi-cellular organisms, the present inventionis not limited to this particular application. In accordance withalternative embodiments, components of biological entities other thannucleic acids may be recovered and purified using the present invention.For example, proteins present within a virus, bacteria, or cell may beexposed through lysis, and then recovered and analyzed and/or purifiedin accordance with embodiments of the present invention.

And while the above description has focused upon microfluidicarchitectures which combine the isolation of components of viruses,cells, or bacteria, with purification of the isolated components, thisis not required by the present invention. In alternative embodiments,isolation and lysis of one or a subset of biological entities from asample may be followed immediately by analysis, without necessarilyrequiring an intervening purification step.

Such alternative embodiments may be particularly valuable wheremicrofluidic isolation of only one or a small number of biologicalentities has been achieved, reducing the overall complexity of theresulting lysed mixture. Such alternative embodiments may also provevaluable where mixtures are analyzed utilizing techniques highlysensitive to a specific target present therein.

While the present invention has been described herein with reference toparticular embodiments thereof, a latitude of modification, variouschanges and substitutions are intended in the foregoing disclosure, andit will be appreciated that in some instances some features of theinvention will be employed without a corresponding use of other featureswithout departing from the scope of the invention as set forth.Therefore, many modifications may be made to adapt a particularsituation or material to the teachings of the invention withoutdeparting from the essential scope and spirit of the present invention.It is intended that the invention not be limited to the particularembodiment disclosed as the best mode contemplated for carrying out thisinvention, but that the invention will include all embodiments andequivalents falling within the scope of the claims.

What is claimed is:
 1. A method for preparing a plurality of cDNAlibraries from single cells in a microfluidic apparatus, wherein theapparatus comprises: (a) a solid substrate; (b) a cell separation andprocessing system embedded in the substrate that includes an arrangementof flow channels configured so that individual cells from a cellpopulation can be singly isolated; (c) one or more inlet channels in thesubstrate connected with the cell separation and processing system sothat a reagent can be delivered through the inlet channel(s) andcombined with cells isolated by the system; and (d) one or more outletchannels in the substrate connected with the cell separation andprocessing system so that contents obtained by lysing isolated cells canbe flowed through the outlet channel(s) and kept separate; wherein themethod comprises: (1) receiving a sample of cells into the cellseparation and processing system of the apparatus; (2) processing thesample in the cell separation and processing system such that aplurality of single cells from the sample are fluidically isolated fromall other cells in the sample, thereby producing isolated single cells;(3) combining a lysing chemical or buffer with the isolated cells suchthat mRNA is liberated from each of at least some of the isolated singlecells in a manner that keeps the mRNA from each of the isolated singlecells separate; and (4) reverse transcribing and amplifying the mRNAliberated in step (3) such that a separate cDNA library is formed fromat least some of the isolated single cells; thereby producing saidplurality of cDNA libraries from single cells.
 2. The method of claim 1,wherein the method further comprises: (5) flowing the separate cDNAlibraries through the outlet channel(s) of the apparatus and keepingseparate at least some of the cDNA libraries formed in step (4).
 3. Themethod of claim 1, wherein step (2) and step (3) are performedseparately.
 4. The method of claim 1, wherein the cell separation andprocessing system is configured to singly isolate cells from the cellpopulation by way of a plurality of valves in the system that areoperable to close channels between single cells.
 5. The method of claim1, wherein the cell separation and processing system is configured tosingly isolate bacteria.
 6. The method of claim 1, wherein the cellseparation and processing system is configured to singly isolateeukaryotic cells.
 7. The method of claim 1, wherein the cell separationand processing system comprises one or more mixing structures configuredto actively mix a lysing agent delivered through the inlet channels withcells singly isolated by the cell separation and processing system. 8.The method of claim 7, wherein the one or more mixing structures arerotary mixers.
 9. The method of claim 1, wherein step (3) comprisesoperating the apparatus so as to actively mix the lysis chemical orbuffer with the single cells.
 10. The method of claim 1, wherein step(3) comprises operating the apparatus so as to diffusively mix the lysischemical or buffer with the single cells.
 11. The method of claim 1,further comprising sequencing cDNA libraries from the single cells. 12.A method for preparing a plurality of nucleic acid libraries from singlecells in a microfluidic apparatus, wherein the apparatus comprises: (a)a solid substrate; (b) a cell separation and processing system embeddedin the substrate that includes an arrangement of flow channelsconfigured so that individual cells from a cell population can be singlyisolated; (c) one or more inlet channels in the substrate connected withthe cell separation and processing system so that a reagent can bedelivered through the inlet channel(s) and combined with cells isolatedby the system; and (d) one or more outlet channels in the substrateconnected with the cell separation and processing system so thatcontents obtained by lysing isolated cells can be flowed through theoutlet channel(s) and kept separate; wherein the method comprises: (1)receiving a sample of cells into the cell separation and processingsystem of the apparatus; (2) processing the sample in the cellseparation and processing system such that a plurality of single cellsfrom the sample are fluidically isolated from all other cells in thesample, thereby producing isolated single cells; (3) combining a lysingchemical or buffer with the isolated cells such that nucleic acid isliberated from each of at least some of the isolated single cells in amanner that keeps the nucleic acid from each of the isolated singlecells separate; and (4) amplifying the nucleic acid liberated in step(3) such that a separate nucleic acid library is formed from at leastsome of the isolated single cells; thereby producing said plurality ofnucleic acid libraries from single cells.
 13. The method of claim 12,wherein the method further comprises: flowing the separate nucleic acidlibraries through the outlet channel(s) of the apparatus and keepingseparate at least some of the nucleic acid libraries formed in step (4).14. The method of claim 12, wherein step (2) and step (3) are performedseparately.
 15. The method of claim 12, wherein the cell separation andprocessing system is configured to singly isolate cells from the cellpopulation by way of a plurality of valves in the system that areoperable to close channels between single cells.
 16. The method of claim12, comprising delivering a lysing agent through the inlet channels andactively mixing the lysing agent with singly isolated cells in the cellseparation and processing system.
 17. The method of claim 16, whereinthe one or more mixing structures are rotary mixers.
 18. The method ofclaim 12, wherein step (3) comprises operating the apparatus so as toactively mix the lysis chemical or buffer with the single cells.
 19. Themethod of claim 12, wherein step (3) comprises operating the apparatusso as to diffusively mix the lysis chemical or buffer with the singlecells.
 20. The method of claim 12, further comprising sequencing nucleicacid or cDNA libraries from the single cells.